Oncolytic adenovirus armed with therapeutic genes

ABSTRACT

The present invention involves compositions and methods for treating cancer using a mutant adenovirus comprising a polynucleotide encoding a therapeutic polypeptide that is targeted to cells with a mutant retinoblastoma pathway. The mutant adenovirus is able to kill the tumor cells without harming cells with a wild type retinoblastoma pathway.

The United States Government owns rights in this invention pursuant tofunding by the National Institutes of Health. This application claimspriority to co-pending U.S. patent application Ser. No. 10/124,608,filed Apr. 17, 2002 and to U.S. Provisional Patent application Ser. No.60/551,932, filed Mar. 10, 2004, each of which is incorporated in theirentirety herein by refernce.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The invention generally relates to the field of oncology and oncolyticadenoviruses. More particularly, it concerns compositions and methods oftreating cancer of the brain in a patient using oncolytic adenovirusesarmed with therapeutic transgenes.

B. Description of Related Art

The development of cancer is understood as the culmination of complex,multistep biological processes, occurring through the accumulation ofgenetic alterations. Many if not all of these alterations involvespecific cellular growth-controlling genes. These genes typically fallinto two categories: proto-oncogenes and tumor suppressor genes.Mutations in genes of both classes generally confer a growth advantageon the cell containing the altered genetic material.

The function of tumor suppressor genes, as opposed to proto-oncogenes,is to antagonize cellular proliferation. When a tumor suppressor gene isinactivated, for example by point mutation or deletion, the cell'sregulatory machinery for controlling growth is upset. The studies ofseveral laboratories have shown that the neoplastic tendencies of suchmutated cells can be suppressed by the addition of a nucleic acidencoding a wild-type tumor suppressor polypepitde (a functional tumorsuppressor) (Levine, 1995).

Mutations and/or loss of function in the retinoblastoma tumor suppressorgene have been associated with tumor formation. In some instances braintumors are metastases to the brain from a primary tumor outside of thecentral nervous system (CNS). Brain tumors derived from metastases aretypically more common than primary tumors of the brain. The most commonprimary tumors that metastasize to the brain are lung, breast, melanoma,and kidney. These brain metastases are usually in multiple sites, butsolitary metastases may also occur.

Gene therapy is a promising treatment for brain tumors including gliomasbecause conventional therapies typically fail and are toxic. Inaddition, the identification of genetic abnormalities contributing tomalignancies is providing crucial molecular genetic information to aidin the design of gene therapies. Genetic abnormalities indicated in theprogression of tumors include the inactivation of tumor suppressor genesand the overexpression of numerous growth factors and oncogenes. Tumortreatment may be accomplished by supplying a polynucleotide encoding atherapeutic polypeptide or other therapeutic that target the mutationsand resultant aberrant physiologies of tumors. It is these mutations andaberrant physiology that distinguishes tumor cells from normal cells. Atumor-selective virus would be a promising tool for gene therapy. Recentadvances in the knowledge of how viruses replicate have been used todesign tumor-selective oncolytic viruses. In gliomas, three kinds ofviruses have been shown to be useful in animal models: reoviruses thatcan replicate selectively in tumors with an activated ras pathway(Coffey et al., 1998); genetically altered herpes simplex viruses(Martuza et al., 1991; Mineta et al., 1995; Andreanski et al., 1997),including those that can be activated by the different expression ofproteins in normal and cancer cells (Chase et al., 1998); and mutantadenoviruses that are unable to express the E1B55 kDa protein and areused to treat p53-mutant tumors (Bischof et al., 1996; Heise et al.,1997; Freytag et al., 1998; Kim et al., 1998). Taken together, thesereports confirm the relevance of oncolytic viruses as anti-canceragents. In all three systems, the goal is the intratumoral spread of thevirus and the ability to selectively kill cancer cells. Geneticallymodified adenoviruses that target cellular pathways at key points haveboth potent and selective anti-cancer effects in gliomas.

Targeting the Rb pathway has noted relevance for the treatment ofgliomas because abnormalities of the p16/Rb/E2F pathway are present inmost gliomas (Fueyo et al., 1998a; Gomez-Manzano et al., 1998).Targeting this pathway by replacement of lost tumor suppressor activitythrough the transfer of p16 and Rb genes has produced cytostatic effects(Fueyo et al., 1998a; Gomez-Manzano et al., 1998). Transfer of E2F-1resulted in powerful anti-cancer effect since the exogenous wild-typeE2F-1 induced apoptosis and inhibited tumor growth in vivo (Fueyo etal., 1998b). However, treating human glioma tumors with existingadenovirus constructs realistically cannot affect significant portionsof the tumor, mainly because replication-deficient adenoviral vectorsare unable to replicate and infect other cells, thus transferring theexogenous nucleic acid to sufficient numbers of cancer cells(Puumalainen et al., 1998). Although targeting the p16/Rb/E2F pathwayproduces an anti-cancer effect in vitro, this imperfection of the vectorsystem limits the therapeutic effect of the gene in vivo.

There is a continued need for additional treatments for cancer,particularly brain tumors, including the creation of additionaloncolytic viruses that are capable of cell-specific replication.Additional treatments include an adenovirus with therapeuticcapabilities or with an ability to be tracked in vivo.

SUMMARY OF THE INVENTION

The present invention provides an oncolytic adenovirus capable ofkilling target cells, such as a tumor cells, with a greater efficiency.The invention takes advantage of the discovery that an adenovirusencoding an E1A polypeptide unable to bind the tumor suppressor proteinRb may not replicate in or kill a cell that has a functional Rb pathway,but may replicate in and kill a cell that has a defective Rb pathway. Invarious aspects of the invention the oncolytic adenovirus is armed orencodes a therapeutic or diagnostic polypeptide. “Armed” is a term thatindicates that the virus contains a heterologous nucleic acid sequenceencoding a polypepitde of interest or a nucleic acid comprising apolynucleotide of interest. In certain embodiments, the nucleic acidencoding a therapeutic polypeptide may encode angiopoietin 2 (Ang-2),humanized yeast cytosine deaminase polypeptide (hyCD) or a sodium-iodidesymporter (NIS) polypeptide. In further embodimens, the NIS polypepitdemay be used in detecting the location of oncolytic adenovirus within asubject. The adenovirus of the present invention can be delivered by anumber routes including, but not limited to intracranial (into the skullcavity) or intravenous administration. The tumor may be a primary tumoror it may be a tumor resulting from a metastasis to the skull or brain.

Embodiments of the invention include an oncolytic adenovirus andreplication defective adenovirus, as well as wildtype adenoviruses.Certain aspects of the invention include an oncolytic adenoviruscomprising an E1A deletion, in particular where the E1A deletion is adeletion of nucleotides encoding amino acids 122 to 129 of the E1Aprotein (Delta 24) and/or Delta-24-300 adenovirus and an expressioncassette encoding a therapeutic or diagnostic gene, including but notlimted to an Ang-2 gene, a yeast cytosine deaminase gene, a humanizedyeast cytosine deaminase gene, and/or a NIS gene. The nucleic acidencoding the yeast cytosine deaminase polypeptide may be a humanizednucleic acid encoding a yeast cytosine deaminase polypeptide. Inpreferred embodiments, the humanized nucleic acid encoding the yeastcytosine deaminase comprises the nucleic acid sequence of SEQ ID NO:5.

An adenovirus of the invention may comprise additional modifications,such as a nucleic acid encoding a modified adenoviral fiber protein,which in certain aspects may comprise a heterologous peptide motif,which targets various proteins expressed on the surface of a cell,including but not limited to adhesion molecules and/or cell surfacereceptors, such as EGFR, EGFRvIII, Tie, and Tie2. The heterologouspeptide motif can be an RGD motif, an EGFR targeting motif, or a Tie2targeting motif. Typically, the targeting motif alters the tropism ofthe virus by providing a chimeric fiber protein that includes theparticular targeting motif.

An adenovirus of the invention, typically, will selectively replicate ina cell having a defective Rb pathway. The defective Rb pathway maycomprise a defective Rb protein or a defect in other proteins that makeup the Rb pathway in a cell. However, some embodiments of the inventionmay be used in conjunction with replication defective adenoviruses orother replication selective, replication competent adenoviruses, orcombinations thereof.

In further embodiments, methods of treating cancer in a patient arecontemplated. The methods include administering to a patient aneffective amount of a composition comprising an oncolytic adenovirus,preferably Delta 24, comprising an expression cassette encodingtherapeutic gene, including but not limited to an Ang-2 gene, a yeastcytosine deaminase gene, a humanized yeast cytosine deaminase and/or aNIS, and administering an effective amount of a pro-drug, wherein thepro-drug is metabolized to a cytotoxic drug by a polypeptide encoded bythe yeast cytosine deaminase gene. The nucleic acid encoding a yeastcytosine deaminase is preferably a humanized nucleic acid encoding ayeast cytosine deaminase. The cancer to be treated may include one ormore cells comprising a mutated Rb pathway. The cancer may comprise oneor more cells comprising a mutated Rb polypeptide. The cell to betreated may be a tumor cell or a brain tumor cell, more particularly aglial cell or glial derived cell. The methods of the invention mayfurther comprise determining whether the cell or cells has a defect,e.g. a mutation, in a gene encoding a polypeptide in the Rb pathway, ina gene encoding Rb or both. A defect may be caused by a deletion or amutation in the nucleic acid encoding a gene, include coding andnon-coding regions of the gene. In certain aspects, a cell is not killedif it does not comprise a mutated polypeptide in the Rb pathway.

The oncolytic adenovirus may be suitably dispersed in apharmacologically acceptable formulation. The composition may comprise asuitable buffer and may further comprise one or more lipids. Thecomposition may be administered through various routes including:intradermal, transdermal, parenteral, intracranial, intravenous,intramuscular, intranasal, subcutaneous, percutaneous, intratracheal,intraperitoneal, intratumoral, perfusion, lavage, direct injection, andoral routes of administration. The compositions may be directly injectedinto a tumor. Furthermore, the administration of a composition may occurmore than once and may be administered at least three times to thepatient. The methods of the invention may further comprise administeringto the patient a second therapy, wherein the second therapy isanti-angiogenic therapy, chemotherapy, immunotherapy, surgery,radiotherapy, immunosuppresive agents, or gene therapy with atherapeutic polynucleotide. The second therapy may be administered tothe patient before, during, after or a combination thereof relative tothe administration of the onclolytic adenovirus composition.Chemotherapy includes, but is not limited to an alkylating agent,mitotic inhibitor, antibiotic, or antimetabolite. The chemotherapy maycomprise administration of CPT-11, temozolomide, or a platin compound.Radiotherapy may include X-ray irradiation, UV-irradiation,γ-irradiation, or microwaves. The oncolytic adenovirus may beadministered to the patient preferably in doses of approximately 10³ toabout 10¹⁵ viral particles; more preferably about 10⁵ to about 10¹²viral particleseven more preferably about 10⁷ to about 10¹⁰ viralparticles.

In still further embodiments, methods include methods for treating abrain tumor in a patient comprising identifying a patient having a braintumor; and contacting the tumor with an oncolytic adenovirus comprisingan expression cassette, preferably an expression cassette comprising anAng-2 gene, a yeast cytosine deaminase gene, and/or a NIS gene. Theoncolytic adenovirus may also comprise a targeting moiety. The methodsmay include contacting the tumor with the adenovirus by injecting theadenovirus intracranially into the patient.

Embodiments of the invention include methods for treating a subjecthaving a brain tumor by determining that a cell in the tumor has amutation in the Rb pathway; administering intracranially to the patientan oncolytic adenovirus comprising an expression cassette, wherein theexpression cassette comprises a therapeutic or diagnostic gene. Themethods may further include administering to the patient a pro-drug thatis converted by cytosine deaminase to a therapeutic drug.

Methods of the invention also include treatment of a subject having atumor that has metastasized to the brain by determining that a cell inthe tumor has a mutation in the Rb pathway; administering intracraniallyto the patient an oncolytic adenovirus and a therapeutic gene such as,but not limited to Ang-2 gene, yeast cytosine deaminase gene, humanizedyeast cytosine deaminase, and/or NIS gene. The methods may furtherinclude administering to the patient a pro-drug that is converted bycytosine deaminase to a therapeutic drug, a therapeutic or ananti-angiogenic agent(s) (e.g., anti-sense VEGF or its equivalents).

Embodiments of the invention also include an adenovirus (e.g., Delta 24,Delta-24-300) comprising; and an expression cassette encoding asodium-iodide symporter (NIS) gene. The nucleic acid encoding NIS maycomprise the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3.Additionally, the adenovirus may further comprise additionalmodifications, such as a nucleic acid encoding a modified adenoviralfiber protein, which may comprise a heterologous peptide motif,preferably the heterologous peptide motif is an RGD, vIII, or PEPHC1motif.

The expression cassette may comprise a chimeric polynucleotide encodingAng-2, hyCD, or NIS and a nucleic encoding a second peptide orpolypeptide, wherein a sequence encoding a protease-cleavable amino acidlinker is between the NIS coding sequence and the nucleic acid encodingthe second peptide or polypeptide. The protease-cleavable amino acidlinker may be an auto-cleaving amino acid sequence. The sequenceencoding a protease-cleavable linker can be fused in-frame to the 3′ or5′ end of the Ang-2, NIS and/or hyCD encoding polynucleotide. In certainaspects, the protease cleavable linker is cleaved by furin. Theprotease-cleavable linker may be identical to a linker present in acytoplasmic protein.

The expression cassette may comprises a chimeric polynucleotidecomprising the Ang-2, NIS or hyCD encoding polynucleotide, orcombination thereof, and a nucleic acid sequence encoding a secondpeptide or polypeptide, wherein the chimeric polynucleotide encode aninternal ribosome entry site between NIS encoding polynucleotide and asecond nucleic sequence encoding a second polypeptide.

Further embodiments of the invention include methods of monitoring thelocation of an oncolytic adenovirus or a nucleic acid encoding theoncolytic adenovirus in a mammal, comprising the steps of administeringto a mammal an oncolytic adenovirus comprising a nucleic sequenceencoding a sodium-iodide symporter (NIS), wherein the expression of theNIS sequence in cells permits cellular uptake of a diagnostic agent;administering to the mammal a diagnostic agent in an amount sufficientto permit transport of the diagnostic agent by the NIS and detection oftransported diagnostic agent; and determining the location of thetransported diagnostic agent in the mammal as an indication of thelocation of the oncolytic adenovirus or nucleic acid encoding theoncolytic adenovirus. The step of detecting can be performedquantitatively to determine the amount of transported diagnostic agentin the mammal. The diagnostic agent may be iodine. Iodine may be iodineradionuclides ¹³¹I, ¹²³I, ¹²⁴I, or ¹²⁵I. The diagnostic agent may alsobe technesium pertechnetate, rhenium perrhenate, radioactive iodine, orother diagnostic elements used in the art. In certain aspects of theinvention, a nucleic acid encoding an oncolytic adenovirus comprises achimeric polynucleotide comprising a nucleic acid sequence encoding NISand a second transgene. A second transgene may encode a secondtherapeutic polypeptide.

Embodiments of the invention include methods of treating cancer in apatient comprising administering to a patient an effective amount of acomposition comprising an oncolytic adenovirus comprising an expressioncassette encoding an Ang-2, NIS or hyCD polynucleotide; andadministering an effective amount of a diagnostic agent, a therapeuticagent a therapy enhancing agent or an anti-angiogenic agent. In certainaspects, the diagnostic agent, the therapeutic agent or the therapyenhancing agent is transported into a cell by a NIS polypeptide encodedby the NIS polynucleotide. The cancer may comprise one or more cellscomprising a mutated polypeptide in the Rb pathway, a mutated Rbpolypeptide or both. The method may further comprise determining whetherone or more cell has a mutation, resulting in a defective protein, in agene encoding a polypeptide in the Rb pathway, in the gene encoding Rb,or both. The method may comprise assaying Rb activity. Rb activity maybe assayed for using an anti-Rb antibody or by determining whether Rb inthe cell inhibits E2F activation of transcription. A cell may be a glialcell and/or a tumor cell.

The methods may include an adenovirus composition suitably dispersed ina pharmacologically acceptable formulation. The composition may comprisea suitable buffer and/or a lipid. Composition may be administered by avariety of routes including intradermal, transdermal, parenteral,intracranial, intravenous, intramuscular, intranasal, subcutaneous,percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion,lavage, direct injection, and oral routes of administration. In certainaspects of the invention the composition is directly injected into atumor. Administration of a composition may occur more than once, twice,three times or more.

The method may further comprising administering to the patient a secondtherapy, wherein the second therapy is anti-angiogenic therapy,chemotherapy, immunotherapy, surgery, radiotherapy, immunosuppresiveagents, or gene therapy with a therapeutic polynucleotide. The secondtherapy may be administered to the patient before, during, after or acombination thereof relative to the administration of the onclolyticadenovirus composition. Chemotherapy includes, but is not limited to analkylating agent, mitotic inhibitor, antibiotic, or antimetabolite. Thechemotherapy may comprise administration of CPT-11, temozolomide, or aplatin compound. Radiotherapy may include X-ray irradiation,UV-irradiation, γ-irradiation, or microwaves. The oncolytic adenovirusmay be administered to the patient preferably in doses of approximately10³ to about 10¹⁵ viral particles; more preferably about 10⁵ to about10¹² viral particleseven more preferably about 10⁷ to about 10¹⁰ viralparticles.

Embodiments discussed in the context of a methods and/or composition ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod may be applied to other methods of the invention as well.

The term “about” refers to the imprecision of determining virus, proteinor other amounts and measures, and is intended to include at least onestandard deviation of error for any particular assay, measure orquantification.

“A” or “an,” as used herein in the specification, may mean one or morethan one. As used herein in the claim(s), when used in conjunction withthe word “comprising,” the words “a” or “an” may mean one or more thanone.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein:

FIG. 1 is a schematic representation of the Δ24 adenovirus. Shown arethe 2 Rb-binding regions of the E1A sequence (hatched boxes). The 24nucleotides that have been deleted and the corresponding amino acidtranslation are indicated.

FIG. 2 shows the effects of Δ-24 in a mouse xenograft intracranialglioma tumor model.

FIG. 3 shows visualization of NIS expression with a transfected U87MGtumor implanted subcutaneously in NuNu mice flanks. A 2-3 mm palpabletumor in the animal on the left is clearly visible after injection with0.2 mCi of 99mTcO4 in 0.1 ml PBS. The animal to the right did not have apalpable tumor. The control animals below do not have hNIS expressingflank tumors.

FIG. 4 shows an MRI of nude mouse glioma xenograft model using U87MGcells and a correlated pathologic section (H&E). The MRI panels fromleft to right show the noncontrast T₁, and Gd-contrast T₁, and T₂sequences. The increase in hematoxylin staining of the tumor can beappreciated in the pathologic sections immediately infereior to theTeflon screw.

FIGS. 5A-5C shows viral inclusion bodies (indicative of activelyreplicating adenovirus), FIG. 5A, staining for late transcribed viralgenes (hexon protein), FIG. 5B and the typical “zonal” spread through atumor, FIG. 5C U87MG xenograft infected with Δ24-RGD incubated with E1Aantibody.

FIG. 6 shows a western blot of the transgene expression of yeast CD inU87MG glioma cells using Δ24 adenovirus: lane 1 U87MG cells unifected;lane 2 Δ24-CD at 1 MOI and 24 h post-infection; lane 3 Δ24-CD at 10 MOIand 24 h post-infection; lane 4 Δ24-CD at 100 MOI and 24 hpost-infection; lanes 5-7 are similar to lanes 2-4 except that they are48 h post-infection instead of 24 h; lane 8 Δ24 adenovirus.

FIG. 7 shows expresion of cytosine deaminase by Δ24-hyCD relative to astable expressing clone. The X-axis demonstrates two cell lines atincreasing MOI from 0.1 to 100 MOI and from 24 to 96 h. The Y axis showsthe increase of expression in logarithmic increase.

FIGS. 8A-8D shows a demonstration of animal whole-mount preparation andautoradiography imaging of NIS expression by ¹⁸⁸Re accumulation. Breasttumor (13762F) bearing rat was injected with the eluent fromW-188/Re-188 generator. Panels shown are of (FIG. 8A) an anesthetizedanimal, (FIG. 8B) whole animal mount gross anatomy, (FIG. 8C)autoradiography and (FIG. 8D) gamma imaging (T=tumor, B=bladder,S=stomach).

FIG. 9 shows the pharmacodynamic studies of animals injected with eluantfrom an W/Re-188 Rhenium generator at 100 μCi. The uptake of ¹⁸⁸ReO₄corresponds to tissues that express NIS (thyroid and stomach). Urine andlater fecal accumulation (at 24 hours) is also observed. Importantly,the organ tumor sites to be examined (brain and lung) do not demonstrateNIS-dependent active accumulation of radionuclides.

FIG. 10 shows accumulation of Tc⁹⁹O₄ in a U251MG glioma cell line aftertransient transfections with the hNIS-containing plasmid (pcDNA3.1-Zeo).Comparing the accumulations of Tc⁹⁹O₄ perechnetate is determined by cpmafter incubation for 30 min with 2 μCi. Cells were then washed withice-cold Hanks balanced salt solution and Tc^(99m)O₄ released by addingabsolute ethanol. Parental cell line was compared to the active clone inthe first two lanes. Controls using the inhibitor to the hNIS pump(NaClO₄) against the parental cell line, vector control and finallypositive clone simultaneousely inhibited by NaClO₄.

FIG. 11 shows accumulations of Tc⁹⁹O₄ in U251 glioma cell line infectedwith Δ24-NIS. Cells incubated with virus for 1.5 h then washedextensively and mixed at different percentages with uninfected U251cells. Uptake assays were then performed at 48 h and values are given asfold increase (ratios) fo Tc⁹⁹O₄ within the cells compared to controlcells. A clear progressive fold increase in the uprake of redionuclideis demonstrated. Additionally, complete inhibition of NIS activity isseen with the addition of sodium perchlorate.

FIGS. 12A-12F shows a specimen from a patient treated with a singleinjection of Ad-p53 as a dose of 3×10¹⁰ vp in 1 ml (level 1). (FIG. 12A)photograph of sugical specimen that was removed en bloc. The injectioncatheter is protruding from the surface of the tumor. (FIG. 12B)formalin fixed tumor blocks. Specimen from A has been cur perpendicularto the catherter. The hole created by the catheter is evident. (FIG.12C) low power view (300×) of same speciment immunostained with antibodyto p53 protein. The hole from the catheter is at the top of thephotomicrogaph. Transfected tumor cells stain darkly and are distributedwithin 5 mm of the injection site. (FIG. 12D) High power view (500×) ofsame section a C demonstrating transfected cells. (FIG. 12E) View ofadjacent section of that shown distrubution as p53 staining. (FIG. 12F)low power (10×) view of cress-section of whole speciment. The catheterwas within the central hole. Blue staining around hole showsdistribution of exogenous p53.

FIGS. 13A-13B shows Generation of Δ24-hyCD adenovirus. (FIG. 13A) Thehumanized yeast cytosine deaminase sequence is depicted as the completenucleotide sequence of yeast CD as well as the nucleotide substitutions(highlighted in bold) for optimized humanized codon preference. A Kozaksequence is placed immediately before the starting codon (italicized).Proximal HindIII and distal XbaI restriction sites are placed betweenparentheses. (FIG. 13B) Schematic illustration of Δ24-hyCD showing the24-bp deletion in the E1A region (nucleotides and corresponding aminoacid residues are shown) and the insertion of the modified cytosinedeaminase (hyCD) expression mini-cassette in the deleted E3 region.

FIGS. 14A-14B shows analyses of the expression and enzymatic activity ofhyCD. (FIG. 14A) Western blot analyses of the expression of exogenoushyCD in U251MG cells. Expression was apparent by 24 h after infection ina dose-dependent manner. As expected, mock (M) or Δ24-treatment at anpfu/cell of 100 did not result in the expression of CD. The expressionlevel of actin is showed as a loading control. (FIG. 14B) Thin layerchromatography analyses of cytosine deaminase. U251MG cells stablytransfected with the hyCD encoding polynucleotide were treated withcytosine at the indicated times and assessed for hyCD activity. Themigrated uracil spot was visualized with ultraviolet excitation at 260λ.Lanes 5 and 6 showed a dose-dependence positive result. Negative(cytosine) and positive (uracil) controls are shown in lanes 1 and 2,respectively.

FIG. 15 shows dose response curves of parental and hyCDstable-transfected U251MG cells treated with 5-FC, as assessed by cellviability assay. Note the shift to the left of the IC₅₀ curve for thehyCD stable-transfected U251MG cells in the presence of 5-FC. IC₅₀values for both cultures are indicated.

FIGS. 16A-16C shows in vitro antiglioma effect of Δ24-hyCD. (FIG. 16A)Crystal violet analyses of the cytopathic effect of Δ24-hyCD or Δ24 inU251MG cells treated with either 5-FU or 5-FC. Each well represents adifferent time period of 5-FU or 5-FC treatment indicated in days. (FIG.16B) Quantification of the viability of U251MG cells by MTT assay,treated with Δ24 (left) or Δ24-hyCD (right) and 5-FC at the indicateddoses. UVi, UV-inactivated adenoviral treatment, 5 MOI. (FIG. 16C)Demonstration of Δ24-hyCD-mediated bystander effect. U25 1 MG cells weretreated with Δ24-hyCD or Δ24 (at an pfu/cell of 10) 24 h afterinfection, and conditioned media was collected. In one set of studies,the conditioned media were inactivated by UV to ensure that noreplication-competent virus was carried over. Conditioned media at theindicated volumes were transferred to fresh U251MG cultures andincubated for 48 h. Viability was assessed by MTT assay.

FIG. 17 shows the treatment with Δ24-hyCD+5-FC significantly improvessurvival of nude mice implanted with U87MG intracranial xenografts. Dataare represented as Kaplan-Meier survival curves from the day of U87MGintracranial implantation (day 0) after intratumoral injection (day 3)with a single dose of experimental viral groups or PBS alone controls.5-FC was administered on day 5 after treatment (E) or on day 15 aftertreatment (L). At both time points, the combination of Δ24-hyCD-mediatedoncolysis and 5-FC effects resulted in a significant increase insurvival compared with vehicle, Δ24 alone, or in combination with 5-FC,or Δ24-hyCD alone. The P value (determined by logrank test) comparedΔ24-hyCD with the combination of Δ24-hyCD and 5-FC. Shown are resultsfrom a representative experiment. The median survival of the animalsreceiving Δ24-hyCD treated early (E=5 days) or late (L=15 days) were notdifferent.

FIGS. 18A-18B. FIG. 18A shows Tie2 expression in glioma cell lines: a)RT-PCR, and b) Western blotting analysis (membrane subfraction) of theexpression of Tie2 in a panel of glioma cell lines. HUVEC cells andNIH3T3 cells were used as positive and negative cotnrols. FIG. 18B showsAng-2 downregulates VEGF secretion. Shown here is the secreted VEGFexpressed as a percentage relative to that of the mock-infected cells(equal to 100%) (ELISA). Values shown as mean±SD (+, P<0.005; *, P>0.5).

FIG. 19 shows Ang-2 reduced expression of HIF-1α in glioma cells.(Western Blot, Nuclear extracts). Protein nuclear levels of HIF-1αdecreased after Ang-2 transfer, compared with control-treated cells innormoxia (21% O2) and hypoxia (1% O2) conditions in U-87 MG and D54 MGcells. Note that the expression of HIF-1α, did not modify after Ang-2transfer into U-251 MG cells, CMV, AdCMV; Ang-2, AdAng-2.

FIG. 20 shows Ang2 downmodulates HIF-DNA binding activity. U-87 MG cellswere treated with AdAng2 (Ang2), AdCMV-pA (CMV) or were mock-infected.Equal amounts of nuclear extracts were analyzed for HIF-DNA bindingactivity in a hypoxic or normoxic setting. Competitive experiments wereperformed using wild-type (wt) or mutant (mut) oligonucleotides. Dataare represented as the mean of three independent experiements (SD wasless than 15%).

FIG. 21 shows Ang-2 inhibits Ang-1 mediated MEK/ERK phosphorylation ofU-87 MG glioma cells. HUVEC and U-87 MG cells were mock-, AdCMV- orAdAng-2-Infected. 24 hours later they were overnights serum-starved, andthen stimulated with rhAng-2 for 10 min. Cell lysates were collected andanalyzed by Western blotting for expression of phospho- and totalp42/p44 MAPK. Ang-1 increased ERK 1/2 phosphorylation what was inhibitedby the viral-transduced Ang-2.

FIG. 22 shows Transcriptional modulation of VEGF by Ang-2 is probablyrelated to the downmodulation of HIF-1α proteins levels (U-87 MG).Luciferase activity is expressed as relative to the VEGF-1.5 kb promoteractivity in mock-treated cells (equal to 100%). The result shows thatAng-2 decreased the transcriptional activity of the two contstructs thatcontain the HIF-1α binding site (2.6 and 1.5 kb promoters; Gomez-Manzanoet al., 2003), however did not modified the activity of the 0.35-kbpromoter, susceptivle to p53NHL regulation.

FIG. 23 is a presentation of the E1A region of wild-type (wt), Delta-24,Delta-300 and Delta-24-300 adenoviruses. Solid area: p300 binding area;hatched area: Rb Binding area.

FIG. 24 shows Anti-glioma effect of Delta-24-300, Delta-24, Delta-300and wt-Ad in vitro. Cellse were infected at indicated MOI and viabilitywas assessed ty Trypan blue exclusion. Data shown as the relativepercentage of cells alive with UVi-infected cultures equal to 100%

FIG. 25 shows Anti-glioma effect of Delta-24-300, Delta-24, Delta-300and wt-Ad in vitro. Cell viability was assessed by crystal violetstaining after viral infection with a range of MOI, 0.5-10 MOI, asindicated in top left panel. M: mock-infection; Uvi. UV-inactivatedwt-Ad.

FIGS. 26A-26B show differential expression of Delta-24-300 viralproteins in glioma and normal human astrocyte (NHA) cultures. (FIG. 26A)Western blot analysis of the E1A and fiber proteins in U-251 MG and NHAcell extracts 16 hours after infection with Delta-24, Delta-300 orDelta-24-300 at an MOI of 50. (FIG. 26B) Actin expression is used as aloading control. Quantification of the fiber protein signal bydensitometry following normalization to actin levels. Fiber levels fromDelta-24-trated cultures are arbitrarily given the value of 100%.

FIGS. 27A-27B. FIG. 27A shows analyses or adenoviral-induced E2F-1promoter activity. Proliferating NHA were transfected with the E2F-1reporter construct, and 1 h later the cells were treated with theindicated adenovirus at 5 MOI. Luciferase activity was determined 20 hafter infection. Data is shown as relative means±SEM of normalizedluciferase measurements (Ad300, equal to 100%). FIG. 27B shows Viralreplication analysis. Replicating NHA and U-87 MG giloma cell lines wereinfected with wt Ad, Delta-24, Delta-300 or Delta-24-300 at 1 MOI. Threedays later viral titers were determined by TCID50 method. Viral titersfrom two independent experiments were normalized to wild-type adenovirustiters. Values in the chart represent the difference between the viraltiters in U-87 MG cells and NHA.

FIG. 28 shows chimeric fiberprotein. Tail: N-terminal of AdS fiberprotein (1-83aa); T4 febritin; bacteriophage T4 fibritin helican domainand fold on (233-487 aa); Linker: G4SG4SG4S linker; Ligand: PEPHC1(HFLIIGFMRRALCGA (SEQ ID NO:19).

FIG. 29 shows infectivity assay of human mesenchymal stem cells. Light(left) and fluorescence (right) microscopy of the same fieldof humanmesenchymal stem cells at the indicated hours after infection with areplication-deficient adenovirus carrying the GFP cDNA (Ad-GFP) or thetropism-modified Ad-GFP adenovirus (Ad-GFP-RGD) at 100 MOI.

FIG. 30 shows MRI studies nude mice bearing intracranial U-87 MGxenografts. Animals were imaged 7 and 14 days after cell implantation.Shown are T2 images (left) and gross morphologic features (right, H&E)of the brains of tumors treated with UV-inactivated Delta-24-300 orDelta-24-300. Note that Delta-24-300 treatment resulted in inhibition ofthe tumoral growth as seen simulteaneously in both pathological andimaging studies.

FIG. 31 shows specific binding of EGFRvIII-peptide (PEPCH1) toEGFRvIII-expressing cells. Briefly, the inventors performed membranefluorescence staining of single-cell suspensions after incubating U-87MG, U87.wtEGFR, and U-87.ΔEGFRvIII (Nishikawa et al., 1994) with theanti-EGFRvIII peptide (PEPCH1)-FITC (1 μM) (Campa et al., 2000) for 30min. The cells were then washed twice with PBS and then analyzed forfluorescence with an EPICS XL-MCL flow cytometer (Beckman-Coulter Inc.,Miami, Fla.) using a 488 nm argon laser for excitation. Fluorescence wastetected with a 520 nm band-pass filter, and all cytometric data wasanalyzed with the System II software (Beckman-Coulter Inc.). Data areshown as the percentages of FICT-positive cells (empty graph) comparedto mock-treated cells (solid graphs)from a representative experiment.

FIG. 32 shows data are shown after normalization of the binding value,as FITC-positive U87.ΔEGFR cells equal to 100%.

FIG. 33 shows construction of pVK.Delta-24-vIII. Schema showing keyinformation of the adenoviral vector pVK500C and accomplished homologousrecombinations. E1A adenoviral gene containing a 24 base-pair deletionthat encompasses an area responsible for binding Rb protein (nucleotides923 to 946), corresponding to the amino acids L₁₂₂TCHEAGF₁₂₉ (asDelta-24, Fueyo et al., 2000), was transfected into E. coli BJ5183,together with pVK500C, previously linearalized with ClaI enzyme. Theresultant plasmid, this time linearilized with SwaI, and the recombinantDNA for the fiber-fibritin-ligand (FFL=DNA sequence for PEPHC1) chimerawas cotransfected into E. coli BJ5183, obtaining pVK.Delta-24-vIII.

FIGS. 34A-34B shows analysis of the Delta-24-vIII genome structure. FIG.34A shows confirmation of the mutant-E1A region. DNA isolated fromDelta-24-vIII adenovirus was subjected to PCR assay utilizing pair ofprimers flanking the deletion in the E1A region and FIG. 34B showsposteriorly subjected to restriction enzyme analysis using BstXI whichcleaves tice in the E1A region amplified from Wild-type adenovirus, andone in the E1A from Delta-24. Two clones were asolited and tested(Clon#12 and #13). pVK500C and pVK.Delta24 were used as controls.

FIGS. 35A-35B Analysis of the Delta-24-vIII genome structure. FIG. 35Ashows confirmation of the chimeric fiber. FIG. 35B shows DNA isolatedfrom Delta-24-vIII adenovirus was subjected to PCR assay utilizing pairof primers as depicted below. Two clones were asolited and tested (Clons#12 and #13). pVK500C and pVK.Delta24 were used as controls.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Malignant tumors that are intrinsically resistant to conventionaltherapies are significant therapeutic challenges. Such malignant tumorsinclude, but are not limited to malignant gliomas and recurrent systemicsolid tumors such as lung cancer. Malignant gliomas are the mostabundant primary brain tumors having an annual incidence of 6.4 casesper 100,000 (CBTRUS, 2002-2003). These neurologically devastating tumorsare the most common subtype of primary brain tumors and are one of thedeadliest human cancers. In the most aggressive cancer, manifestationglioblastoma multiforme (GBM), median survival duration for patientsranges from 9 to 12 months, despite maximum treatment efforts (Hess etal., 1999). A prototypic disease, malignant glioma is inherentlyresistant to current treatment regimens (Shapiro and Shapiro, 1998). Infact, in approximately ⅓ of patients with GBM the tumor will continue togrow despite treatment with radiation and chemotherapy. Median survivaleven with aggressive treatment including surgery, radiation, andchemotherapy is less than 1 year (Schiffer, 1998). Because few goodtreatment options are available for many of these refractory tumors, theexploration of novel and innovative therapeutic approaches is essential.

One potential method to improve treatment is based on the concept thatnaturally occurring viruses can be engineered to produce an oncolyticeffect in tumor cells (Wildner, 2001; Jacotat, 1967; Kim, 2001; Geoergeret al., 2002; Yan et al., 2003; Vile et al., 2002, each of which isincorporated herein by reference). In the case of adenoviruses, specificdeletions within their adenoviral genome can attenuate their ability toreplicate within normal quiescent cells, while they retain the abilityto replicate in tumor cells. One such conditionally replicatingadenovirus, Δ24, has been described by Fueyo et al. (2000), see alsoU.S. Patent Application No. 20030138405, each of which are incorporatedherein by reference. The Δ24 adenovirus is derived from adenovirus type5 (Ad-5) and contains a 24-base-pair deletion within the CR2 portion ofthe E1A gene. Significant antitumor effects of Δ24 have been shown incell culture systems and in malignant glioma xenograft models.

Oncolytic adenoviruses include conditionally replicating adenoviruses(CRADs), such as Delta 24, which have several properties that make themcandidates for use as biotherapeutic agents. One such property is theability to replicate in a permissive cell or tissue, which amplifies theoriginal input dose of the oncolytic virus and helps the agent spread toadjacent tumor cells providing a direct antitumor effect.

Embodiments of the present invention couple the oncolytic component ofDelta 24 with a transgene expression approach to produce an armed Delta24. Armed Delta 24 adenoviruses may be used for producing or enhancingbystander effects within a tumor and/or producing or enhancingdetection/imaging of an oncolytic adenovirus in a patient, or tumorassociated tissue and/or cell. It is contemplated that the combinationof oncolytic adenovirus with various transgene strategies will improvethe therapeutic potential against a variety of refractory tumors, aswell as provide for improved imaging capabilities. In certainembodiments, an oncolytic adenovirus may be administered with areplication defective adenovirus, another oncolytic virus, a replicationcompetent adenovirus, and/or a wildtype adenovirus. Each of which may beadminstered concurrently, before or after the other adenoviruses.

Embodiments of the invention include the Delta 24 adenovirus comprisingan expression cassette containing a heterologous gene. Examples of suchheterologous genes include therapeutic genes, pro-drug convertingenzymes, cytosine deaminase (to convert 5-FC to 5-FU), a yeast cytosinedeaminase, a humanized yeast cytosine deaminase, an image enhancingpolypeptides, a sodium-iodide symporter, anti-sense or ihibitory VEGF,Bcl-2, Ang-2, or interferons alpha, beta or gamma. In certain aspects ofthe present invention, a Delta 24 oncolytic adenoviral strategy iscoupled with an Ang-2 transgene, sodium-iodide symporter (NIS)transgene, humanized yeast CD or a yeast CD transgene approach foraugmenting bystander effects and/or obtaining imaging of the replicatingvirus within an in vivo tumor setting.

II. Armed Oncolytic Adenovirus Δ24

The in vitro and in vivo oncolytic effects of Δ24 adenovirus have beendemonstrated. Generally, adenovirus is a 36 kb, linear, double-strandedDNA virus (Grunhaus and Horwitz, 1992). Adenoviral infection of hostcells results in adenoviral DNA being maintained episomally, whichreduces the potential genotoxicity associated with integrating vectors.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

A. Δ24—Sodium Iodide Symporter (NIS)

Embodiments of the invention include a Δ24 adenovirus encoding an NISpolypeptide. NIS is a transmembrane pump that was originally isolatedfrom the thyroid gland and functions physiologically by concentratingiodide within thyroid tissue. NIS is also able to mediate the uptake andconcentration of other diagnostic and therapeutically importantradionuclides, such as Technicium-99m pertechnetate (^(99m)TcO₄) andRhenium-188 perrenate (¹⁸⁸ReO₄). By combining a tumor-specific oncolyticadenovirus with expression of the NIS polypeptide radioisotopes may beconcentrated in the presence of viral replication to allow superiorimaging of an infected tumor while potentially augmenting therapeuticeffectivness.

The mechanism mediating iodide uptake across the basloateral membrane ofthyroid follicular cells has been elucidated by cloning andcharacterization of the sodium iodide symporter (NIS) (Smanik et al.,1996; Dai et al., 1996). The human NIS encoding polynucleotide containsan open reading frame of 1,928 nucleotides and encodes a 643-amino acidtrans-membrane protein, which has an expected molecular weight of 69kDa. This pump has been shown to be capable of concentrating multipleisotopes within the intracellular compartment, including ¹²³I, ¹²⁵I and¹³¹I. NIS is an intrinsic membrane glycoprotein with 13 putativetransmembrane domains which is responsible for the ability of cells ofthe thyroid gland to transport and sequester iodide. An NIS of thepresent invention is comprised of a polypeptide having the activity of asodium iodide symporter, including, but not limited to polypeptides ofSEQ ID NO:2 and 4 that are encoded by the nucleic acid sequences of SEQID NO:1 and 3, for human and rat respectively. NIS expression in thyroidtissues is dependent upon stimulation of the cells by pituitary-derivedthyroid stimulating hormone (TSH) and can therefore be readilysuppressed in this tissue by treatment with Thyroxine. TSH-regulated NISexpression is specific for thyroid cells, whereas many other organs donot concentrate iodine due to lack of NIS expression. Cloning andcharacterization of the human and rat NIS genes (SEQ ID NO:1 and SEQ IDNO:3 respectively; GenBank Accession numbers AC005796 and U60282respectively) permits NIS encoding nucleic acid delivery intonon-thyroid cells, thereby allowing these cells to trap and sequesterradio-labeled iodine.

According to the present invention, NIS functions well as a localizationtag for several reasons. The NIS is synthesized in the mammal, using themammals own protein synthetic machinery, and thus is recognized as self,thereby avoiding a potential immune response. Furthermore, the NIS is auseful localization tag according to the present invention as it shouldhave no significant effect on the biological properties of thegenetically modified cells. Given that the only known function of theNIS is to transport iodine across the cell membrane, it should notadversely affect endogenous cellular function (U.S. Pat. No. 6,586,411,which is incorporated herein by reference).

The therapeutic index of successful treatment of thyroid carcinomas andtheir metastases by ¹³¹I is based on the expression of the NIS withinthyrocytes (Levy et al., 1998a; Kohrle, 1999; Levy, 1998b).Additionally, treatment failure parallels the loss of expression of NIS.Because radioisotopes are widely established in diagnostic nuclearradiographic studies as well as being useful in treatment strategies, aNIS encoding polynucleotide is an attractive target forradioisotope-mediated cancer gene therapy (La Perle et al., 2002).Recently, many tumor lines, including glioma cell lines, have beengenetically modified to express NIS using both viral and nonviraltransfer vectors (Cho et al., 2000 and 2002). All related studies hadthe concerns of complete vector delivery and specific tumor targeting.If NIS expression within cancer cells can approach or exceed itsexpression in typical thyroid tumor cells then accumulation of ¹³¹I canresult in tissue ablative doses of ionizing radiation. This interstitialdosing approximates brachytherapy. In addition, very short-livedisotopes, such as ^(99m)TCO₄ (T_(1/2) 6 hours), which is a commonradioisotope tracer used in clinical practice, can easily be used todemonstrate the expression of NIS within a tumor focus. These features,coupled with the delivery features of Δ24, make this combination anattractive and powerful system that can be used both to image and treattumors.

1. Expression of NIS in Tumors

A number of reports have demonstrated the uptake of variousradionuclides in tumor models using the NIS pump. The human and the ratform of NIS have each been cloned and transfected into various tumorcell lines by multiple authors (e.g., Haberkorn, 2001; Cho et al., 2002;Cho et al.., 2000; Haberkorn et al., 2001; Haberkorn and Altman, 2002).It is unknown whether this protein could effectively engage itselfappropriately into the membrane of tumor cells. In fact, Eskandar et al.(1991) demonstrated that rat NIS (rNIS) could be expressed in Xenopuslaevis oocytes. It was shown that this pump was sodium dependent andcapable of transporting a wide, variety of anions (I⁻, CIO⁻, SCN⁻,SeCN⁻, NO₃ ⁻, Br⁻, BF₄ ⁻, IO₄ ⁻;, and BrO₃ ⁻), although, perchlorate(ClO₄) inhibited the NIS pump. Cho et al. (2002) expressed hNlS in humanglioma cells via transfection with a replication-deficient adenovirus,producing accumulation within mouse xenograft tumors and demonstratingaccretion of ¹²⁵I and ^(99m)Tc-pertechnetate. The uptake of ¹²⁵I wasapproximately 20-fold higher for tumors expressing hNlS compared withnon-expressing tumors. Similar results were demonstrated by Haberkornand Altmann (2001, 2002) who found that an hNIS-expressing hepatoma wasable to concentrate ¹³¹I as demonstrated by gamma camera imaging.Although different authors have shown the ability of NIS to concentratevarious isotopes within a tumor expressing this pump, the lack ofadequate and efficient delivery mechanisms is a problem that continuesto plague investigators.

The compositions and methods described herein are designed to improvetransgene expression by combining the power of an oncolytic virus withNIS expression for radionuclide accumulation.

B. Δ24—Cytosine Deaminase (CD)

Cytosine deaminase (CD, EC 3.5.4.1) catalyzes the hydrolysis of cytosineto uracil. The enzyme, which plays an important role in microbialpyrimidine metabolism (O'Donovan and Neuhard, 1970), has been isolatedfrom several different microorganisms, but does not appear to be presentin mammalian cells (Nishiyama et al., 1985).

The physical properties of CD from various organisms have been shown todiffer significantly in terms of molecular weight, stability, andsubunit composition. For example, CD from Salmonella typhimurium hasbeen purified to homogeneity (by SDS-PAGE) and is composed of 4 subunitsof 54 kilodaltons (kDa) each (West et al., 1982) while the enzyme fromEscherichia coli has a molecular weight of 200 kDa and is composed of 35and 46 kDa subunits (Katsuragi et al., 1986). Both of these enzymes arehighly thermostable, and maintain high activity at 55° C.

Bakers' yeast (Saccharomyces cerevisiae) has also been used as a sourcefor yeast CD. CD previously obtained from Saccharomyces cerevisiae has amolecular weight of 34 kDa as determined by gel filtration (Ipata etal., 1971, 1978) and 32-33 kDa as determined by SDS-PAGE and amino acidanalysis (Yergatian et al., 1977). The CD enzyme that has beenpreviously isolated from bakers' yeast therefore appears to be amonomeric protein. In certain embodiments a humanized yeast cytosinedeaminase (hyCD) (SEQ ID NO:5 and 6) may be used minimize any immunereactions to the protein.

Solutions of previously isolated bakers' yeast CD maintain activity forat least 48 hr when stored at 4° C. between pH 5-9 (Ipata et al., 1971,1978). However, at 37° C., a crude preparation of bakers' yeast CD hasbeen shown to lose half of its activity in 1 hr, and a purified form ofthe enzyme has a half-life of 30 min (Katsuragi, 1987). Thus, thethermal instability of CD from bakers' yeast, along with its lowmolecular weight, distinguish it from the bacterial enzymes describedearlier.

CD has been used therapeutically for the conversion of the pro-drug5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU)(Katsuragi et al., 1987; Nishiyama et al., 1985; Senter et al., 1991).However, bacterial sources of CD are impractical for such use, requiringlarge-scale cultivation in order to obtain adequate activity.

Yeast can be used as a source of CD to overcome these problems. However,the thermal instability of the previous yeast-derived product requiresthat the enzyme be immobilized prior to its use (Katsuragi et al.,1987). Thus, the isolation and purification of a thermally stable yeastCD provides an improved enzyme for use in anticancer therapy. Such novelconstructs increase the efficiency or usefulness of the enzyme inanticancer therapy (U.S. Pat. No. 5,545,548, which is incorporatedherein by reference).

1. Δ24 Delivery of Cytosine Deaminase

To illustrate Δ24's ability to function as an effective delivery vectorfor the expression of exogenous genes and its potent augmentation of theoncolytic effect, high functional levels of cytosine deaminase wereexpressed in the setting of a Δ24 infection. The Δ24 backbone wasmodified using a yeast cytosine deaminase encoding polynucleotide clonedinto the E3 region of Δ24. Derived from yeast, the cytosine deaminasegene has superior catalytic properties compared with the bacterial formof the enzyme (Kievit, 1999). Extremely high specific activity of thisenzyme has been detected when the oncolytic virus is allowed to infecthuman glioma tumors. Assays have demonstrated a highly specificconversion of cytosine to uracil or 5-fluorocytosine (5-FC) to 5FU.

Additionally, preliminary studies using a MTT cell viability assay inU251MG and U87MG glioma cell lines reveal clear and separable cellkilling based on a pro-drug bystander effect after infection with theΔ24 oncolytic virus containing the cytosine deaminase gene. Onedifficulty encountered in these early studies has been that theoncolytic properties of Δ24 are so powerful that it is difficult todistinguish the effects of viral replication and cell lysis fromconcomitant bystander effects (Wei et al., 1995). To date, theimprovement seen in glioma cell killing appears to occur in a muchshorter amount of time using the pro-drug-converting enzyme comparedwith using Δ24 alone. This finding is consistent with the notion thatconversion to a cytotoxic compound (5-FC to 5-FU) can diffuse into alarger number of cells to more quickly kill the cells. These resultsappear to suggest that gene-dependent enzyme/pro-drug therapy (GDEPT)increases the oncolytic potency of the virus and serves as proof ofconcept that exogenous genes can be delivered using oncolytic adenoviralconstructs.

C. Adenoviral Delivery of Anti-Angiogenics

Angiogenesis is critical for the development and maintenance ofglioblastomas, the most malignant and common form of primary braintumors. Current evidence indicates that recruitment of tumor vesselsfrom the normal surrounding tissue requires a delicate balance betweenthe timing and level of expression of two major angiogenesis factors:angiopoietin 2 (Ang-2) and the vascular endothelial growth factor(VEGF). Ang-2 is typically expressed at sites of vascular remodeling inthe adult, notably in the female reproductive tract. Detailedlocalization of Ang-2 in the ovary by in situ hybridization revealedthat in regions of active vascular remodeling it was either expressedtogether with VEGF at sites of vessel sprouting and ingrowth, or in theabsence of VEGF at sites of frank vessel regression. These expressionpatterns led to the proposal that Ang-2 plays a facilitative role atsites of vascular remodeling in the adult by blocking constitutivestabilizing actions. Further, it is suggested that such destabilizationby Ang-2 in the presence of high VEGF levels primes the vessels to mounta robust angiogenic response. However, such destabilization by Ang-2 inthe absence of VEGF is instead proposed to lead to frank vesselregression. Thus, hypothetically, overexpression of Ang-2 in gliomasshould result in the formation of poorly differentiated and inefficienttumor vessels that will undergo regressive changes overriding vesselproliferation. Therefore a maintained overexpression of Ang-2 in gliomasshould have potent anti-glioma effect.

Combining oncolysis with anti-angiogenesis may produce a synergisticeffect since the anticancer mechanisms are different but complementary.In addition, the success of the strategy is assisted by the relativelyslow rhythm of oncolytic propagation. That allows time for ananti-angiogenic nucleic acid or polypeptide, sucy as, but not limted toan Ang-2 protein, to be produced, ultimately favoring delivery to theextracellular compartment. For that reason, the oncolytic adenovirus isused as an improved adenoviral vector to deliver high and continuouslevels of Ang-2 to the tumor. Another use of replication-competentadenoviruses that are currently being pursued is applyingreplication-competent systems as facilitators for the delivery ofreplication-deficient E1-deleted adenovirus vectors. This is a usefulapproach because co-infection of a cancer cell with both areplication-deficient E1-deleted adenovirus and a replication-competentadenovirus results in the replication of both adenoviruses. This isbecause the replication-deficient vector uses the expression of the E1Aprotein by the replication-competent adenovirus to replicate. Thissystem can thus generate and multiply the number of copies of exogenousprotein. The inventors will use this strategy to simultaneously deliveran anti-angiogenic nucleic acid or polypeptide, such as Ang-2 (in areplication competent, oncolytic or replication defective adenovirus)and antisense VEGF (in a replication competent, oncolytic or replicationdefective adenovirus) (Im et al., 1999) adenoviruses to human gliomaxenografts. In certain aspects of the invention a targeted ornon-targeted gene therapy approach with Delta-24 adenovirus will be used(Fueyo et al., 2002 and 2003).

The effects of Delta-24 based antiangiogenic agents may be compared toor used in conjunction with other antiangiogenesis agents that are orwill be tested in clinical trials for patients with malignant gliomas.Second, in certain aspects, the inventors are using cell lines with agreater clinical relevance, as LN229 (SNB19 as alternative). These celllines exhibit an invasive phenotype when implanted intracranialy inanimal models. Both cell lines express the Tie2 receptor and aretumorigenic in intracranial settings. Finally, since it has beenreported that E1A downregulates HIF-1α activity (Zoltan et al., 1996),methods and compositins are contemplated that will differentiateAng-2-mediated regulation of HIF-1 a and the regulation that adenoviralE1A exerts on the HIF-1α transcription factor.

Despite indications that VEGF regulates Ang-2 in tumors, there is notany evidence of a feedback signaling loop which will be required tocoordinate and modulate the expression of the Ang-2 and VEGF molecules.The specific time when the expression of Ang-2 is required during thedifferent stages of tumor formation is also not known. Finally, notreatments have been developed on the basis of dysregulation of theputative feedback loop and the consequent induction of an imbalancecaused by the expression of Ang-2 together with the downregulation ofVEGF.

The inventors put forth the idea that there is a regulatory signalingloop involving the coordinated and sequential expression of VEGF andAng-2, and elucidating the underlying mechanisms can lead to developinga better model for the angiogenic process that occurs in human gliomas,as well development of improved compositions and methods or thetreatment of such. Moreover, exploiting the interdependence between VEGFand Ang-2 could be used to develop more effective and rationalanti-angiogenesis therapies for brain tumors than currently exist.

The inventors use oncolytic adenoviruses for the simultaneous targetingof Ang-2 or Ang-2 and VEGF (the inhibition or down regulation). Theinventors also plan on characterizing the interaction between Ang-2 andVEGF; and the role of Ang-2 expression in a dynamic tumor model ofglioma angiogenesis. The inventors will obtain efficacious preclinicalevidence for supporting the translation of their studies to the clinicfor single and/or combination anti-angiogenesis treatments for malignantgliomas.

Angiogenesis refers to vessel formation by remodeling the primaryvascular network or by sprouting from existing vessels (reviewed inYancopoulos et al., 2000). The “angiogenesis switch” is “off” when theeffect of pro-angiogenic molecules is balanced by the activity ofanti-angiogenic molecules, and is “on” when the net balance between themolecules is tipped in favor of angiogenesis (reviewed in Carmeliet andJain, 2000). Angiogenesis has an essential role in the development andmaintenance of solid tumors, including malignant gliomas.

Malignant gliomas, the most common subtype of primary brain tumors, areaggressive, highly invasive, and neurologically destructive tumorsconsidered to be among the deadliest of human cancers. In its mostaggressive manifestation, glioblastoma multiforme (GBM), median survivalranges from 9 to 12 months, despite maximum treatment efforts.

The striking and dramatic induction of angiogenesis in GBM has fueledthe speculation that progression to GBM requires activation ofangiogenesis and has stimulated significant efforts in the developmentof agents that will block this process. Two pathways in particular havereceived considerable attention. They are vascular endothelial growthfactor (VEGF) and Angiopoietin 2. VEGF has been shown to be critical forthe earliest stages of vasculogenesis, promoting endothelial cellproliferation, differentiation, migration, and tubular formation. Genetargeting studies have shown that a deficiency of VEGF or VEGFreceptors, Flt-1, or Flk-1, results in early embryonic lethality causedby defects in angiogenesis and vasculogenesis (reviewed in Yancopouloset al., 2000, Ferrara 2002). Specific VEGF inhibitors have recently beenintroduced into clinical glioma trials, and results are forthcoming.Encouragingly, the putative antiangiogenic agent, thalidomide, has beenshown to have activity in patients with recurrent high-grade gliomas(Fine et al., 2000). VEGF inhibitors may include VEGF-neutralizingchimeric proteins such as soluble VEGF receptors. (See Aiello, PNAS, 92,10457 (1995)). In particular, they may be VEGF-receptor-IgG chimericproteins. Another VEGF inhibitor contemplated for use in the presentinvention is antisense phosphorothio oligodeoxynucleotides (PS-ODNs).Examples of anti-angiogenesis agents include, but are not limited to,retinoid acid and derivatives thereof, 2-methoxyestradiol, ANGIOSTATIN®protein, ENDOSTATIN® protein, suramin, squalamine, tissue inhibitor ofmetalloproteinase-I, tissue inhibitor of metalloproteinase-2,plasminogen activator inhibitor-1, plasminogen activator inhibitor-2,cartilage-derived inhibitor, paclitaxel, platelet factor 4, protaminesulphate (clupeine), sulphated chitin derivatives (prepared from queencrab shells), sulphated polysaccharide peptidoglycan complex (sp-pg),staurosporine, modulators of matrix metabolism, including for example,proline analogs ((1-azetidine-2-carboxylic acid (LACA),cishydroxyproline, d,1-3,4-dehydroproline, thiaproline], α,α-dipyridyl,β-aminopropionitrile fuimarate,4-propyl-5-(4-pyridinyl)-2(3h)-oxazolone; methotrexate, mitoxantrone,heparin, interferons, 2 macroglobulin-serum, chimp-3, chymostatin,beta.- cyclodextrin tetradecasulfate, eponemycin; fumagillin, goldsodium thiomalate, d-penicillamine (CDPT), β-1-anticollagenase-serum,α-2-antiplasmin, bisantrene, lobenzarit disodium,n-(2-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”,thalidomide; angostatic steroid, cargboxynaminolmidazole;metalloproteinase inhibitors such as BB94. Other anti-angiogenesisagents include antibodies, preferably monoclonal antibodies againstthese angiogenic growth factors: bFGF, aFGF, FGF-5, VEGF isoforms,VEGF-C, HGF/SF and Ang-1/Ang-2. (Ferrara and Alitalo (1999) NatureMedicine 5:1359-1364. Calbiochem (San Diego, Calif.) carries a varietyof angiogensis inhibitors including (catalog number/product name)658553/AG 1433; 129876/Amiloride, Hydrochloride; 164602/Aminopeptidase NInhibitor; 175580/Angiogenesis Inhibitor; 175602/Angiogenin (108-123);175610/Angiogenin Inhibitor; 176600/Angiopoietin-2, His·Tag®, Human,Recombinant, Mouse, Biotin Conjugate; 176705/Angiostatin K1-3, Human;176706/Angiostatin K1-5, Human; 176700/Angiostatin(& Protein, Human;178278/Apigenin; 189400/Aurintricarboxylic Acid; 199500/Benzopurpurin B;211875/Captopril; 218775/Castanospermine, Castanospermum australe;251400/D609, Potassium Salt; 251600 Daidzein;288500/DL-a-Difluoromethylomithine, Hydrochloride; 324743/Endostatin™Protein, His·Tag®, Mouse, Recombinant, Spodoptera frugiperda;324746/Endostatin™ Protein, Human, Recombinant, Pichia pastoris;324733/Endostatin™ Protein, Mouse, Recombinant, Pichia pastoris; 329740Eriochrome® Black T Reagent; 344845 Fumagillin, Aspergillus fumigatus;345834 Genistein; 375670/Herbimycin A, Streptomyces sp.;390900/4-Hydroxyphenylretinamide; 407293/a-Interferon, Mouse,Recombinant, E. coli; 407306/g-Interferon, Human, Recombinant, E. coli;05-23-3700/Laminin Pentapeptide; 05-23-3701/Laminin Pentapeptide Amide;428150/Lavendustin A; 454180/2-Methoxyestradiol; 475838/Mifepristone;475843/Minocycline, Hydrochloride; 4801/Neomycin Sulfate;521726/Platelet Factor 4, Human Platelets; 553400/Radicicol,Diheterospora chlamydosporia; 554994/RHC-80267; 565850/Shikonin;573117/SMC Proliferation Inhibitor-2w; 572888/SU1498; 572632/SU5614;574625/Suramin, Sodium Salt; 608050/TAS-301; 585970/(±)-Thalidomide;605225/Thrombospondin, Human Platelets; 616400/Tranilast;654100/TSRI265; 676496/VEGF Inhibitor, CBO-P11; 676493/VEGF Inhibitor,Flt2-11; 676494 VEGF Inhibitor, Je-11; 676495 VEGF Inhibitor, V1;676480NEGF Receptor 2 Kinase Inhibitor I; 676485/VEGF Receptor 2 KinaseInhibitor II; 676475/VEGF Receptor Tyrosine Kinase Inhibitor, and othersuch agents known to those of ordinary skill in the medical arts.

In the case of the angiopoeitins, genetic studies in mice have shownthat angiopoietin-1 (Ang-1) promotes remodeling and stabilization ofVEGF-induced vessels (Suri et al.. 1996; Suri et al., 1998) throughinteractions between endothelial cells and surrounding pericytes and theextracellular matrix. Ang-2 appears to be a natural antagonist of Ang-1,responsible for destabilizing mature vessels in the context of vesselregression or angiogenesis in a VEGF-dependent manner (Maisonpierre etal., 1997). Consequently, Ang-2 expression results in reversal of thematuration process mediated by Ang-1, leading to a disruption ofinteractions between endothelial cells, pericytes, and the extracellularmatrix. In the presence of VEGF activity, the Ang-2-mediated effect isfollowed by sprouting and ingrowth of new vessels (i.e.,neovascularization). Conversely, in the absence of concomitant VEGFexpression, Ang-2-mediated destabilization leads to the regression ofblood vessels (Holash et al., 1999). On the basis of these results, theinventors envision a therapeutic strategy targeted to the tumor in whichAng-2 is induced in the setting of VEGF inhibition, a combinationpredicted to cause vascular collapse in the tumors. The strong geneticvalidation has justified ongoing angiopoietin-directed drug developmentinitiatives.

Although the mechanism driving the angiogenic burst in GBM has yet to beelucidated, existing evidence points to a pivotal role for VEGF(Millauer et al. 1994; Cheng et al. 1996). It has long been held thatthe rapid proliferative rate of GBM creates local ischemia and hypoxia,leading to a VEGF-mediated induction of angiogenesis. This hypothesis isbased on the observation that marked VEGF expression is localized toregions of perinecrotic (palisading) cells at expanding edges of thetumor (Plate et al. 1992). However, a recent study has shed doubt onthis theory. Holash et al. (1999) and Zagzag et al. (2000) demonstratedthat glioma cells implanted into murine brain actually home initially toexisting vasculature. This co-optation of host vessels eventually leadsto the induction of Ang-2 expression in the host endothelial cells andAng-2-mediated destabilization (as manifested by the lifting ofastrocytic foot processes away from endothelial cells and disruption ofthe normal pericyte cuffing) and subsequent regression of existingvessels and necrosis. Tumor cell necrosis resulting from vascularregression and hypoxia appear to trigger the expression of VEGF and theonset of angiogenesis. These intriguing findings support the view that akey mechanism in early tumor angiogenesis may be the direct effect oftumor cells on existing blood vessels rather than the orthodox view ofnew blood vessel growth into the hypoxemic, growing tumor bed.

The role of Tie2 in angiogenesis has been demonstrated in studies thathave involved the deletion of this receptor. Specifically, Tie2 knockoutmice die early in development because of immature blood vessels and lackof vessel development, (Sato et al., 1995). Ligands for the Tie2receptor include Ang-1 and Ang-2 (Davis et al., 1996; Maisonpierre etal., 1997). Ang-1 phosphorylates Tie2 in cultured endothelial cells,whereas Ang-2 is a naturally occurring antagonist of Ang-1, competingwith Ang-I for binding to Tie2 (Maisonpierre et al., 1997). In solidtumors, newly formed tumor vessels are often tenuous, poorlydifferentiated and undergo regressive changes even as blood vesselproliferation continues. The failure of many solid tumors to form awell-differentiated and stable vasculature could indeed be attributableto the fact that newly formed tumor vessels continue to overexpressAng-2. Thus, a persistent blockade of Tie2 signaling may prevent vesseldifferentiation and maturation, contributing to the generally tenuousand leaky quality of tumor vessels. Inhibition of Tie2 using a solublereceptor resulted in inhibition of tumor angiogenesis (Lin et al.,1998;Zadeh et al., 2004). Although, it was thought that Tie2 is avascular-specific receptor, preliminary evidence suggests that theexpression of Tie2 is not limited to endothelial cells (Valable et al.,2003; Poncet et al., (2003). Importantly, cells of neuroectodermicorigin express Tie2 (Valable et al., 2003). Therefore, the negativeregulation of the Tie2 receptor and the growth proliferationtransduction signal that its activation triggers can be used as ananti-proliferation target in cells other than endothelial cells thatexpress Tie2, including cancer cells.

Tumor angiogenesis begins when tumor cells release molecules that sendsignals to the surrounding normal host tissue. Such signaling activatesspecific genes in the host tissue that, in turn, generate proteins thatencourage the growth of new blood vessels. Among these molecules, twoproteins appear to be the most important for sustaining tumor growth:VEGF and Ang-2. Imbalances in the coordinated timing and expression ofthese molecules lead to vessel regression. The confirmation of theAng-2-mediated downmodulation of HIF is underscored by the observationthat the overexpression of HIF-1α has been associated with increasedpatient mortality in several cancer types. Moreover, in preclinicalstudies, inhibiting the activity of HIF-1 has marked effects on tumorgrowth. This approach may yield important mechanistic information aboutthe abnormal regulation of angiogenesis in gliomas. Characterizing anactive Tie2 pathway in glioma cells will have an enormous scientific andclinical relevancy. This description together with the explanation ofthe overall role of Tie2 role in cell proliferation will define a noveltarget for glioma therapy, one that will permit therapies to besimultaneously directed against the glioma cell mass as well as theangiogenic vascular network. Furthermore, the data obtained from thesestudies will provide a rational basis for the development of a phaseI/II clinical trial to assess the toxicity and efficacy of a tripletreatment for malignant gliomas, one that will combine theoverexpression of Ang-2 a decrease in the effect VEGF, and theproduction of oncolysis.

D. Therapeutic Genes

Aspects of the invention include nucleic acids or genes that encode adetectable and/or therapeutic polypeptide. In certain embodiments of thepresent invention, the gene is a therapeutic, or therapeutic gene. A“therapeutic gene” is a gene which can be administered to a subject forthe purpose of treating or preventing a disease. For example, atherapeutic gene can be a gene administered to a subject for treatmentor prevention of diabetes or cancer. Examples of therapeutic genesinclude, but are not limited to, Rb, CFTR, p16, p21, p27, p57, p73,C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II,BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF, G-CSF, thymidine kinase,mda7, fus, interferon α, interferon β, interferon γ, ADP, p53, ABLI,BLC1, BLC6, CBFA1, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS2, ETV6, FGR,FOX, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1,MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3, YES, MADH4, RB1, TP53, WT1,TNF, BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, ApoAI, ApoAIV,ApoE, Rap1A, cytosine deaminase, Fab, ScFv, BRCA2, zac1, ATM, HIC-1,DPC-4, FHIT, PTEN, ING1, NOEY1, NOEY2, OVCA1, MADR2, 53BP2, IRF-1, Rb,zac1, DBCCR-1, rks-3, COX-1, TFPI, PGS, Dp, E2F, ras, myc, neu, raf,erb, fms, trk, ret, gsp, hst, abl, E1A, p300, VEGF, FGF, thrombospondin,BAI-1, GDAIF, or MCC.

In certain embodiments of the present invention, the therapeutic gene isa tumor suppressor gene. A tumor suppressor gene is a gene that, whenpresent in a cell, reduces the tumorigenicity, malignancy, orhyperproliferative phenotype of the cell. This definition includes boththe full length nucleic acid sequence of the tumor suppressor gene, aswell as non-full length sequences of any length derived from the fulllength sequences. It being further understood that the sequence includesthe degenerate codons of the native sequence or sequences which may beintroduced to provide codon preference in a specific host cell.

Examples of tumor suppressor nucleic acids within this definitioninclude, but are not limited to APC, CYLD, HIN-1, KRAS2b, p16, p19, p21,p27, p27mt, p53, p57, p73, PTEN, Rb, Uteroglobin, Skp2, BRCA-1, BRCA-2,CHK2, CDKN2A, DCC, DPC4, MADR2/JV18, MEN1, MEN2, MTS1, NF1, NF2, VHL,WRN, WT1, CFTR, C-CAM, CTS-1, zac1, scFV, MMAC1, FCC, MCC, Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), 101F6, Gene 21 (NPRL2), or a gene encoding a SEM A3polypeptide and FUS1. Other exemplary tumor suppressor genes aredescribed in a database of tumor suppressor genes atwww.cise.ufl.edu/˜yy1/HTML-TSGDB/Homepage.html. This database is hereinspecifically incorporated by reference into this and all other sectionsof the present application. Nucleic acids encoding tumor suppressorgenes, as discussed above, include tumor suppressor genes, or nucleicacids derived therefrom (e.g., cDNAs, cRNAs, mRNAs, and subsequencesthereof encoding active fragments of the respective tumor suppressoramino acid sequences), as well as vectors comprising these sequences.One of ordinary skill in the art would be familiar with tumor suppressorgenes that can be applied in the present invention.

In certain embodiments of the present invention, the therapeutic gene isa gene that induces apoptosis (i.e., a pro-apoptotic gene). A“pro-apoptotic gene amino acid sequence” refers to a polypeptide that,when present in a cell, induces or promotes apoptosis. The presentinvention contemplates inclusion of any pro-apoptotic gene known tothose of ordinary skill in the art. Exemplary pro-apoptotic genesinclude CD95, caspase-3, Bax, Bag-1, CRADD, TSSC3, bax, hid, Bak, MKP-7,PERP, bad, bcl-2, MST1, bbc3, Sax, BIK, BID, and mda7. One of ordinaryskill in the art would be familiar with pro-apoptotic genes, and othersuch genes not specifically set forth herein that can be applied in themethods and compositions of the present invention.

The therapeutic gene can also be a gene encoding a cytokine. The term‘cytokine’ is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators. A“cytokine” refers to a polypeptide that, when present in a cell,maintains some or all of the function of a cytokine. This definitionincludes full-length as well as non-full length sequences of any lengthderived from the full length sequences. It being further understood, asdiscussed above, that the sequence includes the degenerate codons of thenative sequence or sequences which may be introduced to provide codonpreference in a specific host cell.

Examples of such cytokines are lymphokines, monokines, growth factorsand traditional polypeptide hormones. Included among the cytokines aregrowth hormones such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-24 LIF, G-CSF,GM-CSF, M- CSF, EPO, kit-ligand or FLT-3.

Other examples of therapeutic genes include genes encoding enzymes.Examples include, but are not limited to, ACP desaturase, an ACPhydroxylase, an ADP-glucose pyrophorylase, an ATPase, an alcoholdehydrogenase, an amylase, an amyloglucosidase, a catalase, a cellulase,a cyclooxygenase, a decarboxylase, a dextrinase, an esterase, a DNApolymerase, an RNA polymerase, a hyaluron synthase, a galactosidase, aglucanase, a glucose oxidase, a GTPase, a helicase, a hemicellulase, ahyaluronidase, an integrase, an invertase, an isomerase, a kinase, alactase, a lipase, a lipoxygenase, a lyase, a lysozyme, apectinesterase, a peroxidase, a phosphatase, a phospholipase, aphosphorylase, a polygalacturonase, a proteinase, a peptidease, apullanase, a recombinase, a reverse transcriptase, a topoisomerase, axylanase, a reporter gene, an interleukin, or a cytokine.

Further examples of therapeutic genes include the gene encodingcarbamoyl synthetase I, omithine transcarbamylase, arginosuccinatesynthetase, arginosuccinate lyase, arginase, fumarylacetoacetatehydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin,glucose-6-phosphatase, low-density-lipoprotein receptor, porphobilinogendeaminase, factor VIII, factor IX, cystathione beta.-synthase, branchedchain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase,propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoAdehydrogenase, insulin, beta.-glucosidase, pyruvate carboxylase, hepaticphosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein,T-protein, Menkes disease copper-transporting ATPase, Wilson's diseasecopper-transporting ATPase, cytosine deaminase, hypoxanthine-guaninephosphoribosyltransferase, galactose-1-phosphate uridyltransferase,phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase,α-L-iduronidase, glucose-6-phosphate dehydrogenase, HSV thymidinekinase, or human thymidine kinase.

Therapeutic genes also include genes encoding hormones. Examplesinclude, but are not limited to, genes encoding growth hormone,prolactin, placental lactogen, luteinizing hormone, follicle-stimulatinghormone, chorionic gonadotropin, thyroid-stimulating hormone, leptin,adrenocorticotropin, angiotensin I, angiotensin II, β-endorphin,β-melanocyte stimulating hormone, cholecystokinin, endothelin I,galanin, gastric inhibitory peptide, glucagon, insulin, lipotropins,neurophysins, somatostatin, calcitonin, calcitonin gene related peptide,β-calcitonin gene related peptide, hypercalcemia of malignancy factor,parathyroid hormone-related protein, parathyroid hormone-relatedprotein, glucagon-like peptide, pancreastatin, pancreatic peptide,peptide YY, PHM, secretin, vasoactive intestinal peptide, oxytocin,vasopressin, vasotocin, enkephalinamide, metorphinamide, alphamelanocyte stimulating hormone, atrial natriuretic factor, amylin,amyloid P component, corticotropin releasing hormone, growth hormonereleasing factor, luteinizing hormone-releasing hormone, neuropeptide Y,substance K, substance P, or thyrotropin releasing hormone.

As will be understood by those in the art, the term “therapeutic gene”includes genomic sequences, cDNA sequences, and smaller engineered genesegments that express, or may be adapted to express, proteins,polypeptides, domains, peptides, fusion proteins, and mutants. Thenucleic acid molecule encoding a therapeutic gene may comprise acontiguous nucleic acid sequence of about 5 to about 12000 or morenucleotides, nucleosides, or base pairs.

E. Mechanism of Δ24 Oncolytic Virus

A dramatic increase in the cellular proliferation that is characteristicof the transformation from low-grade to intermediate-grade glioma is inlarge part related to dysregulation of the p16/Rb/E2F pathway (Fueyo etal., 2000; Fueyo et al., 1998; Chintala, 1997). Most compelling is thelack of mutational overlap seen among the various members of thispathway, which argues that an important therapeutic advance in thetreatment of these tumors could be achieved by specifically targetingthe Rb pathway (Kyritsis and Yung, 1996; Fueyo et al., 1999). DisruptedRb status will likely provide opportunities to utilize agents thatoperate exclusively in Rb -deficient tumor cells (Fueyo et al., 1999).Most normal human brain cells are usually quiescent. Cells in thecentral nervous system (CNS) rarely divide, and these cells arespecifically triggered to divide in a limited fashion. Tight regulatorycontrols have evolved which strictly limit cells from undergoing celldivision. The p16/Rb/E2F pathway is an important pathway for maintainingthe non-dividing status of fully differentiated cell or negativelyregulates the cell-cycle progression of dividing normal cells.

Human adenovirus normally infects human cells, which are quiescent(nondividing) or dividing cells (normal or cancer cells). Uponintroduction of this virus into a human cell (viral infection), theadenovirus DNA is immediately transcribed by the synthesis of E1Aadenoviral protein. The CR2 region of E1A protein interacts specificallywith Rb protein and leads to release of E2F, forcing cell entry intoS-phase (the DNA Synthesis phase) of the cell cycle and maintaining thecell in the dividing cycle. This series of events effectivelycommandeers the host cell exclusively for the purpose of expressingvirally encoded proteins. Active production of adenoviral particlesdepends on this ability to drive cells into an active mode ofreplication, a critical feature of oncolytic viruses. As a consequenceof their biologic characteristics, tumor cells provide a replicatingenvironment that favors such activity. Mutations in critical sequencesof the viral genome render the adenovirus unable to bind to andinactivate tumor suppressor proteins. These modified adenoviruses areable to replicate exclusively in cells lacking a functional target tumorsuppressor gene (tumor cells only).

Thus, the expression of an E1A protein with a 24 base pair deletion inthe CR2 region prevents the protein from binding to and inactivating Rb.This attenuated E1A-mutant adenovirus is unable to replicate withinnormal quiescent cells that have a funtionally active Rb pathway. Incontrast, tumor cells are permissive to viral replication, which in turnefficiently invade and lyse human glioma cells both in vitro and invivo.

The oncolytic potential of Δ24 is dramatic compared with otherconditionally replication-deficient adenoviruses, such as Onyx-015. Theeffects of Δ24 in a mouse xenograft intracranial glioma tumor model areshown in FIG. 2. In this case, the curve representing RA55 carries thedeletion in the E1B region as in Onyx-015. The oncolytic adenovirus doesnot have the same degree of potency as Δ24 at comparable doses used (inthis case 1×10⁸ pfu). Also shown is the negative control Δ24 that isinactivated by ultraviolet exposure. The antitumor effects of Δ24 havebeen demonstrated in various human tumor cell lines and in animalxenograft models with known defects of the p16/Rb/E2F pathway.Permissive replication of Δ24 in cell lines with p16/Rb/E2F defects iscontrasted with the highly attenuated replication in normal astrocytesand normal quiescent fibroblasts. Additionally, the activity of thisvirus is attenuated when introduced into tumor cells in which Rb hasbeen functionally restored through stable or transient transfectiontechniques.

Several factors favor the use of oncolytic adenoviruses for thetreatment of brain tumors. First, gliomas do not metastasize, andtherefore an efficient local approach should be enough to cure thedisease. Second, every glioma harbors several populations of cellsexpressing different genetic abnormalities (Sidransky et al., 1992;Collins and James, 1993; Furnari et al., 1995; Kyritsis et al., 1996).Thus, the spectrum of tumors sensitive to the transfer of a single geneto cancer cells may be limited. Third, replication competentadenoviruses can infect and destroy cancer cells that are arrested inG₀. Since gliomas invariably include non-cycling cells, this property isimportant. Finally, the p16-Rb pathway is abnormal in the majority ofgliomas (Hamel et al., 1993; Henson et al., 1994; Hirvonen et al., 1994;Jen et al., 1994; Schmidt et al., 1994; Costello et al., 1996; Fueyo etal., 1996b; Kyritsis et al., 1996; Ueki et al., 1996; Costello et al.,1997), thus making the Δ24 strategy appropriate for most of thesetumors. Although the loss of the retinoblastoma tumor suppressor genefunction has been associated with the causes of various types of tumorsand is not limited to treatment of gliomas.

In other embodiments of the invention, an E1A mutation (e.g., a Δ24mutation in E1A) may be used in combination with mutations in the E1Bregion of the same adenovirus, thus producing a double mutantadenovirus. In certain embodiments of the invention an adenovirus maycomprise a Δ24 mutation and a deletion in the E1B region that preventsexpression or function of the E1B55 kD protein. The E1B55 kD protein hasbeen shown to bind to and inactivate p53. The E1B region mutation mayinclude a deletion of adenovirus sequences from 2426 bp to 3328 bp ofgenebank accession number NC-001406, which is incorporated herein byreference.

In certain embodiments of the invention, an oncolytic adenovirus may beused as an adenovirus expression vector. “Adenovirus expression vector”is meant to include those vectors containing adenovirus sequencessufficient to (a) support packaging of the vector and (b) to express apolynucleotide that has been cloned therein. The insertion position of apolynucleotide encoding a heterologous polypeptide of interest withinthe adenovirus sequences is not critical to the invention. Thepolynucleotide encoding the polypeptide of interest may be inserted inlieu of the deleted E3 region in E3 replacement vectors as described byKarlsson et al., (1986) or other region that are not essential for viralreplication in the target cell. Traditional methods for the generationof adenoviral particles is co-transfection followed by subsequent invivo recombination of a shuttle plasmid and an adenoviral helper plasmidinto either 293 or 911 cells (Introgene, The Netherlands).

If an adenovirus has been mutated so that it is unable to replicate oris conditionally replicative (replication-competent under certainconditions), a helper cell may be required for viral replication. Whenrequired, helper cell lines may be derived from human cells such ashuman embryonic kidney cells, muscle cells, hematopoietic cells or otherhuman embryonic mesenchymal or epithelial cells. Alternatively, thehelper cells may be derived from the cells of other mammalian speciesthat are permissive for human adenovirus. Such cells include, forexample Vero cells or other monkey embryonic mesenchymal or epithelialcells. As preferred helper cell line is 293. Various methods ofculturing host and helper cells may be found in the art, for exampleRacher et al., 1995. In the present invention, the adenovirus istypically replication-competent in cells with a mutant Rb pathway. Aftertransfection, adenoviral plaques are isolated from the agarose overlaidcells and the viral particles are expanded for analysis. For detailedprotocols the skilled artisan is referred to Graham and Prevac, 1991.

Alternative technologies for the generation of adenovirus or adenovirusexpression vectors include utilization of the bacterial artificialchromosome (BAC) system, in vivo bacterial recombination in arecA+bacterial strain utilizing two plasmids containing complementaryadenoviral sequences, and the yeast artificial chromosome (YAC) system(PCT publications 95/27071 and 96/33280, which are incorporated hereinby reference).

The nature of the adenovirus vector is not believed to be crucial to thesuccessful practice of the invention. The adenovirus may be of any ofthe 42 different known serotypes or subgroups A-F. Adenovirus type 5 isthe preferred starting material for use in the present invention.Adenovirus type 5 is a human adenovirus about which a great deal ofbiochemical and genetic information is known, and it has historicallybeen used for most constructions employing adenovirus as a vector.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters (e.g., 10⁹-10¹¹ plaque-forming units (pfu) per ml), and they arehighly infective. The life cycle of adenovirus does not requireintegration into the host cell genome.

F. Modifications of Oncolytic Adenovirus

Modifications of oncolytic adenovirus described herein may be made toimprove the ability of the oncolytic adenovirus to treat cancer. Thepresent invention also includes any modification of oncolytic adenovirusthat improves the ability of the adenovirus to treat neoplastic cells.Included are modifications to oncolytic adenovirus genome in order toenhance the ability of the adenovirus to infect and replicate in cancercells by altering the receptor binding molecules.

The absence or the presence of low levels of the coxsackievirus andadenovirus receptor (CAR) on several tumor types can limit the efficacyof the oncolytic adenovirus. Various peptide motifs may be added to thefiber knob, for instance an RGD motif (RGD sequences mimic the normalligands of cell surface integrins), Tat motif, poly-lysine motif, NGRmotif, CTT motif, CNGRL motif, CPRECES motif or a strept-tag motif(Rouslahti and Rajotte, 2000). A motif can be inserted into the HI loopof the adenovirus fiber protein. Modifying the capsid allowsCAR-independent target cell infection. This allows higher replication,more efficient infection, and increased lysis of tumor cells (Suzuki etal., 2001, incorporated herein by reference). Peptide sequences thatbind specific human glioma receptors such as EGFR or uPR may also beadded. Specific receptors found exclusively or preferentially on thesurface of cancer cells may used as a target for adenoviral binding andinfection, such as EGFRvIII.

Cell surface receptors are attractive candidates for the targetedtherapy of cancer. Growth factors and their receptors play importantroles in the regulation of cell division, development, anddifferentiation. Among those receptors, EGFR was the first to beidentified as amplified and/or rearranged in malignant gliomas. EGFRgene amplification in gliomas is often accompanied by generearrangement, resulting in deletions of the coding region. The mostcommon variant, de2-7 EGFR or EGFRvIII, is characterized by an in-framedeletion of 801-bp spanning exons 2-7 of the coding sequence. Thistruncation removes 267 amino acids from the extracellular domain,producing a unique junctional peptide, and renders EGFR unable to bindany known ligand. EGFRvIII is expressed on the cell surface and containsa new tumor-specific protein sequence in its extracellular domain(Sugawa et al. 1990; Ekstrand et al. 1992). The frequency of theEGFRvIII expression in human gliomas is around 20 to 40% (Frederick etal. 2000). Several strategies have already been tested as means forbinding the EGFRvIII receptor using peptides and antibodies. A peptide(PEPHC1) has been synthesized and tested for binding to EGFRvIII andEGFR (Campa et al., 2000, which is incorporated herein by reference inits entirety). In in vitro assays, PEPHC1 bound the recombinant EGFRvIIIextracellular domain or full-length EGFRvIII (solubilized from cellmembranes) in preference to native EGFR. Monoclonal antibodies have beendeveloped with specific activity against this mutant receptor (Lorimeret al. 1996). These antibodies are internalized into the cell afterreceptor binding. Therefore, this receptor is a desirable target foradenoviral tropism since the receptor-binding molecules are efficientlyinternalized and the mutant form offers the opportunity to developtumor-selective targeting strategies.

Although none of the reported adenovirus strategies use the EGFRvIIIreceptor for adenoviral anchorage and internalization, several reportshave characterized EGFR as a potential target in cancer cells. In thesestudies, the adenoviruses redirected to EGFR were more efficient (insome cases by more than 100 fold) and more selective than theadenoviruses using untargeted vectors to infect and transduce cancercells. One of the systems relevant to this proposal uses our Delta-24system in combination with EGFR targeting. In this study, Curiel's group(Hemminki et al. 2001) constructed an adenovirus expressing a secretoryadaptor capable of retargeting the adenovirus to EGFR, resulting in amore than 150-fold increase in gene transfer. A replication-competentdual-virus system secreting the adaptor displayed increased oncolyticpotency in vitro and therapeutic gain in vivo.

Lack of expression in normal cells and achievable targeting usingpeptides and antibodies make the EGFR and EGFRvIII systems very suitablefor the development of targeted oncolytic adenoviruses with hightherapeutic indices (Kuan et al., 2001).

III. RB Pathway

Rb is a tumor suppressor gene whose loss of function is associated withtumor formation. Retinoblastoma protein or Rb, as used herein, refers tothe polypeptide encoded by the retinoblastoma gene (Rb). Theretinoblastoma gene is located at 13q14 in humans and encodes a proteinof approximately 110 kiloDaltons (kD). Unphosphorylated Rb inhibits cellproliferation by sequestering transcription factors (e.g., E2F) andarresting cells in G₁ of the cell cycle. Transcription factors arereleased from Rb when Rb is phosphorylated. The binding of E1A to Rbcauses transcriptional factor release in much the same manner asphosphorylation. Several viral oncoproteins target Rb for inactivationin order to facilitate viral replication. These proteins includeadenovirus E1A, SV40 large T antigen, and papillomavirus E7.

The E1A protein is one of the first virus-specific polypeptidessynthesized after adenoviral infection and is required for viralreplication to occur (Dyson and Harlow, 1992; Flint and Shenk, 1997).Interaction of the Rb protein and the E1A protein results in release ofE2F from pre-existing cellular E2F-Rb complexes. E2F is then free toactivate transcription from E2 promoters of adenovirus and E2F regulatedgenes of an infected cell. The transcriptional activation of thesecellular genes in turn helps to create an environment suitable for viralDNA synthesis in otherwise quiescent cells (Nevins, 1992). Two segmentsof E1A are important for binding Rb; one includes amino acids 30-60 andthe other amino acids 120-127 (Whyte et al., 1988; Whyte et al., 1989).Deletion of either region prevents the formation of detectable E1A/Rbcomplexes in vitro and in vivo (Whyte et al., 1989).

An adenovirus containing a Delta 24 mutation produces an E1A proteinthat cannot bind Rb, causing an infected cell to remain in G₀. Thus amutant Rb pathway and a mutant E1A, along with E2F activation arenecessary for Δ24 adenoviral transcription.

Retinoblastoma (Rb) pathway, as used herein, refers the interaction of agroup of regulatory proteins that interact with Rb or other proteinsthat interact with Rb in regulating cell proliferation (for review seeKaelin, 1999). Proteins within the Rb pathway include, but are notlimited to, Rb, the E2F family of transcription factors, DRTF, RIZ286,MyoD287, c-Ab1288, MDM2289, hBRG1/hBRM, p16, p107, p130, c-Abl tyrosinekinase and proteins with conserved LXCXE motifs, cyclin E-cdk 2, andcyclin D-cdk 4/6. Phosphorylation of Rb releases E2F, which is bound tounphosphorylated Rb. E2F stimulates cyclin E transcription and activity,which results in more Rb phosphorylation. Unphosphorylated Rb acts as atumor suppressor by binding to regulatory proteins that increase DNAreplicaiton, such as E2F (The Genetic Basis of Human Cancer, Vogelsteinand Kinzler eds., 1998).

Defective retinoblastoma pathway, as used herein, refers toinactivation, mutation, or deletion of the Rb or the inability of theupstream or downstream regulatory proteins that interact with Rb toregulate cell proliferation due to a mutation or modification of one ormore proteins, protein activities, or protein-protein interactions.Mutations causing a defective Rb pathway include, but are not limited toinactivating mutations in Rb, INK4 proteins, and CIP/KIP and activatingmutations in the cyclin genes, such as cyclin D/cdk 4, 6 and cyclin E,cdk 2. Mutations in one or another element of the Rb regulatory pathway,including p16, cyclin D, cdk4, E2F or Rb itself, may be mutated inalmost 100 percent of human tumors (The Genetic Basis of Human Cancer,1998). Rb associated tumors include gliomas, sarcomas, tumors of thelung, breast, ovary, cervix, pancreas, stomach, colon, skin, larynx,bladder and prostate.

IV. Methods for Treating Hyperproliferative Conditions

The present invention involves the treatment of hyperproliferativecells, preferably a cell with a disrupted Rb pathway. It is contemplatedthat a wide variety of tumors may be treated using the methods andcompositions of the invention, including gliomas, sarcomas, lung, ovary,breast, cervix, pancreas, stomach, colon, skin, larynx, bladder,prostate, and/or brain metastases, as well as pre-cancerous cells,metaplasias, dysplasias, or hyperplasia.

The term “glioma” refers to a tumor originating in the neuroglia of thebrain or spinal cord. Gliomas are derived form the glial cell types suchas astrocytes and oligodendrocytes, thus gliomas include astrocytomasand oligodendrogliomas, as well as anaplastic gliomas, glioblastomas,and ependymomas. Astrocytomas and ependymomas can occur in all areas ofthe brain and spinal cord in both children and adults.Oligodendrogliomas typically occur in the cerebral hemispheres ofadults. Gliomas account for 75% of brain tumors in pediatrics and 45% ofbrain tumors in adults. The remaining percentages of brain tumors aremeningiomas, ependymomas, pineal region tumors, choroid plexus tumors,neuroepithelial tumors, embryonal tumors, peripheral neuroblastictumors, tumors of cranial nerves, tumors of the hemopoietic system, germcell tumors, and tumors of the sellar region.

Various embodiments of the present invention deal with the treatment ofdisease states comprised of cells that are deficient in the Rb and/orp53 pathway. In particular, the present invention is directed at thetreatment of diseases, including but not limited to retinoblastomas,gliomas, sarcomas, tumors of lung, ovary, cervix, pancreas, stomach,colon, skin, larynx, breast, prostate and metastases thereof.

There are various categories of brain tumors. Glioblastoma multiforme isthe most common malignant primary brain tumor of adults. More than halfof these tumors have abnormalities in genes involved in cell cyclecontrol. Often there is a deletion in the CDKN2A or a loss of expressionof the retinoblastoma gene. Other types of brain tumors includeastrocytomas, oligodendrogliomas, ependymomas, medulloblastomas,meningiomas and schwannomas.

In many contexts, it is not necessary that the cell be killed or inducedto undergo cell death or “apoptosis.” Rather, to accomplish a meaningfultreatment, all that is required is that the tumor growth be slowed tosome degree. It may be that the cell's growth is completely blocked orthat some tumor regression is achieved. Clinical terms such as“remission” and “reduction of tumor” burden also are contemplated giventheir normal usage.

The term “therapeutic benefit” refers to anything that promotes orenhances the well-being of the subject with respect to the medicaltreatment of his/her condition, which includes treatment of pre-cancer,cancer, and hyperproliferative diseases. A list of nonexhaustiveexamples of this includes extension of the subject's life by any periodof time, decrease or delay in the neoplastic development of the disease,decrease in hyperproliferation, reduction in tumor growth, delay ofmetastases, reduction in cancer cell or tumor cell proliferation rate,and a decrease in pain to the subject that can be attributed to thesubject's condition.

A. Adenoviral Therapies

Those of skill in the art are well aware of how to apply adenoviraldelivery to in vivo and ex vivo situations. For viral vectors, onegenerally will prepare a viral vector stock. Depending on the kind ofvirus and the titer attainable, one will deliver 1 to 100, 10 to 50,100-1000, or up to 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰,1×10¹¹, or 1×10¹² infectious particles to the patient in apharmaceutically acceptable composition as discussed below.

Various routes are contemplated for various tumor types. Where discretetumor mass, or solid tumor, may be identified, a variety of direct,local and regional approaches may be taken. For example, the tumor maybe directly injected with the adenovirus. A tumor bed may be treatedprior to, during or after resection and/or other treatment(s). Followingresection or other treatment(s), one generally will deliver theadenovirus by a catheter having access to the tumor or the residualtumor site following surgery. One may utilize the tumor vasculature tointroduce the vector into the tumor by injecting a supporting vein orartery. A more distal blood supply route also may be utilized.

The method of treating cancer includes treatment of a tumor as well astreatment of the region near or around the tumor. In this application,the term “residual tumor site” indicates an area that is adjacent to atumor. This area may include body cavities in which the tumor lies, aswell as cells and tissue that are next to the tumor.

B. Formulations and Routes of Administration to Patients

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions in a form appropriate for theintended application. Generally, this will entail preparing compositionsthat are essentially free of pyrogens, as well as other impurities thatcould be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Aqueous compositions of the present invention comprise an effectiveamount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well know inthe art. Except insofar as any conventional media or agent isincompatible with the present invention, its use in therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. The routes ofadministration will vary, naturally, with the location and nature of thelesion, and include, e.g., intradermal, transdermal, parenteral,intracranial, intravenous, intramuscular, intranasal, subcutaneous,percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion,lavage, direct injection, and oral administration and formulation.Preferred embodiments include intracranial or intravenousadministration. Administration may be by injection or infusion, seeKruse et al. (1994), specifically incorporated by reference, for methodsof performing intracranial administration. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions.

An effective amount of the therapeutic agent is determined based on theintended goal, for example, elimination of tumor cells. The term “unitdose” refers to physically discrete units suitable for use in a subject,each unit containing a predetermined-quantity of the therapeuticcomposition calculated to produce the desired responses, discussedabove, in association with its administration, i.e., the appropriateroute and treatment regimen. The quantity to be administered, bothaccording to number of treatments and unit dose, depends on the subjectto be treated, the state of the subject and the protection desired.Precise amounts of the therapeutic composition also depend on thejudgment of the practitioner and are peculiar to each individual. Theengineered viruses of the present invention may be administered directlyinto animals, or alternatively, administered to cells that aresubsequently administered to animals.

As used herein, the term in vitro administration refers to manipulationsperformed on cells removed from an animal, including, but not limitedto, cells in culture. The term ex vivo administration refers to cellsthat have been manipulated in vitro, and are subsequently administeredto a living animal. The term in vivo administration includes allmanipulations performed on cells within an animal. In certain aspects ofthe present invention, the compositions may be administered either invitro, ex vivo, or in vivo. An example of in vivo administrationincludes direct injection of tumors with the instant compositions byintracranial administration to selectively kill tumor cells.

Intratumoral injection, or injection into the tumor vasculature isspecifically contemplated for discrete, solid, accessible tumorsincluding tumor exposed during surgery. Local, regional or systemicadministration also may be appropriate. For tumors 1.5 to 5 cm indiameter, the injection volume will be 1 to 3 cc, preferably 3 cc. Fortumors greater than 5 cm in diameter, the injection volume will be 4 to10 cc, preferably 5 cc. Multiple injections delivered as single dosecomprise about 0.1 to about 0.5 ml volumes, preferable 0.2 ml. The viralparticles may advantageously be contacted by administering multipleinjections to the tumor, spaced at approximately 1 cm intervals.

In the case of surgical intervention, the present invention may be usedpreoperatively, to render an inoperable tumor subject to resection.Alternatively, the present invention may be used at the time of surgery,and/or thereafter, to treat residual or metastatic disease. For example,a resected tumor bed may be injected or perfused with a formulationcomprising the adenovirus. The perfusion may be continuedpost-resection, for example, by leaving a catheter implanted at the siteof the surgery. Periodic post-surgical treatment also is envisioned.

Continuous administration, preferably via syringe or catheterization,also may be applied where appropriate, for example, where a tumor isexcised and the tumor bed is treated to eliminate residual, microscopicdisease. Such continuous perfusion may take place for a period fromabout 1-2 hr, to about 2-6 hr, to about 6-12 hr, to about 12-24 hr, toabout 1-2 days, to about 1-2 wk or longer following the initiation oftreatment. Generally, the dose of the therapeutic composition viacontinuous perfusion will be equivalent to that given by a single ormultiple injections, adjusted over a period of time during which theperfusion occurs. It is further contemplated that limb perfusion may beused to administer therapeutic compositions of the present invention,particularly in the treatment of melanomas and sarcomas.

Treatment regimens may vary as well, and often depend on tumor type,tumor location, disease progression, and health and age of the patient.Obviously, certain types of tumor will require more aggressivetreatment, while at the same time, certain patients cannot tolerate moretaxing protocols. The clinician will be best suited to make suchdecisions based on the known efficacy and toxicity (if any) of thetherapeutic formulations.

The adenovirus also may be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersionsalso can be prepared in glycerol, liquid polyethylene glycols, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms.

The therapeutic compositions of the present invention are advantageouslyadministered in the form of injectable compositions either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Thesepreparations also may be emulsified. A typical composition for suchpurpose comprises a pharmaceutically acceptable carrier. For instance,the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg ofhuman serum albumin per milliliter of phosphate buffered saline. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil and injectable organic esters such asethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodium chlorideor Ringer's dextrose. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobial agents, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components the pharmaceutical composition are adjusted accordingto well known parameters. When the route is topical, the form may be acream, ointment, or salve.

In a further embodiment of the invention, an adenovirus or a nucleicacid encoding an adenovirus may be delivered to cells using liposome orimmunoliposome delivery. The adenovirus or nucleic acid encoding anadenovirus may be entrapped in a liposome or lipid formulation.Liposomes may be targeted to neoplasic cell by attaching antibodies tothe liposome that bind specifically to a cell surface marker on theneoplastic cell. Liposomes are vesicular structures characterized by aphospholipid bilayer membrane and an inner aqueous medium. Multilamellarliposomes have multiple lipid layers separated by aqueous medium. Theyform spontaneously when phospholipids are suspended in an excess ofaqueous solution. The lipid components undergo self-rearrangement beforethe formation of closed structures and entrap water and dissolvedsolutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Alsocontemplated is a nucleic acid construct complexed with Lipofectamine(Gibco BRL).

C. Combination Therapy

Tumor cell resistance to various therapies represents a major problem inclinical oncology. One goal of current cancer research is to find waysto improve the efficacy of chemo- and radiotherapy, as well as otherconventional cancer therapies. One way is by combining such traditionaltherapies with oncolytic adenovirus therapy. Traditional therapy totreat cancers may include removal of all or part of the affected organ,external beam irradiation, xenon arc and argon laser photocoagulation,cryotherapy, immunotherapy and chemotherapy. The choice of treatment isdependent on multiple factors, such as, 1) multifocal or unifocaldisease, 2) site and size of the tumor, 3) metastasis of the disease, 4)age of the patient or 5) histopathologic findings (The Genetic Basis ofHuman Cancer, 1998).

In the context of the present invention, it is contemplated thatadenoviral therapy could be used in conjunction with anti-cancer agents,including chemo- or radiotherapeutic intervention, as well asradiodiagnositc techniques. It also may prove effective to combineoncolytic virus therapy with immunotherapy.

A “target” cell contacting a mutant oncolytic virus and optionally atleast one other agent may kill cells, inhibit cell growth, inhibitmetastasis, inhibit angiogenesis or otherwise reverse or reduce ahyperproliferative phenotype of target cells. These compositions wouldbe provided in a combined amount effective to kill or inhibitproliferation of the target cell. This process may involve contactingthe cells with the expression construct and the agent(s) or factor(s) atthe same or different times. This may be achieved by contacting the cellwith a single composition or pharmacological formulation that includesboth agents, or by contacting the cell with two distinct compositions orformulations, wherein one composition includes the oncolytic adenvirusand the other includes the second agent.

Oncolytic adenoviral therapy may also be combined withimmunosuppression. The immunosuppression may be performed as describedin WO 96/12406, which is incorporated herein by reference. Examples ofimmunosuppressive agents include cyclosporine, FK506, cyclophosphamide,and methotrexate.

Alternatively, an oncolytic adenovirus treatment may precede or followthe second agent or treatment by intervals ranging from minutes toweeks. In embodiments where the second agent and oncolytic adenovirusare applied separately to the cell, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the second agent and oncolytic adenovirus wouldstill be able to exert an advantageously combined effect on the cell. Insuch instances, it is contemplated that one would contact the cell withboth modalities within about 12-24 hr of each other and, morepreferably, within about 6-12 hr of each other, with a delay time ofonly about 12 hours being most preferred. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

It also is conceivable that more than one administration of eitheroncolytic adenovirus and/or the second agent will be desired. Variouscombinations may be employed, where oncolytic adenovirus is “A” and theother agent is “B”, as exemplified below: A/B/A   B/A/B  B/B/A A/A/BB/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/AB/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations are contemplated. Again, to achieve cell killing,both agents are delivered to a cell in a combined amount effective tokill the cell.

Agents or factors suitable for use in a combined therapy are anyanti-angiogenic agent and/or any chemical compound or treatment methodwith anticancer activity; therefore, the term “anticancer agent” that isused throughout this application refers to an agent with anticanceractivity. These compounds or methods include alkylating agents,topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNAantimetabolites, DNA antimetabolites, antimitotic agents, as well as DNAdamaging agents, which induce DNA damage when applied to a cell.

Examples of chemotherapy drugs and pro-drugs include, CPT11,temozolomide, platin compounds and pro-drugs such as 5-FC. Examples ofalkylating agents include, inter alia, chloroambucil, cis-platinum,cyclodisone, flurodopan, methyl CCNU, piperazinedione, teroxirone.Topoisomerase I inhibitors encompass compounds such as camptothecin andcamptothecin derivatives, as well as morpholinodoxorubicin. Doxorubicin,pyrazoloacridine, mitoxantrone, and rubidazone are illustrations oftopoisomerase II inhibitors. RNA/DNA antimetabolites includeL-alanosine, 5-fluoraouracil, aminopterin derivatives, methotrexate, andpyrazoflirin; while the DNA antimetabolite group encompasses, forexample, ara-C, guanozole, hydroxyurea, thiopurine. Typical antimitoticagents are colchicine, rhizoxin, taxol, and vinblastine sulfate. Otheragents and factors include radiation and waves that induce DNA damagesuch as, γ-irradiation, X-rays, UV-irradiation, microwaves, electronicemissions, and the like. A variety of anti-cancer agents, also describedas “chemotherapeutic agents,” function to induce DNA damage, all ofwhich are intended to be of use in the combined treatment methodsdisclosed herein. Chemotherapeutic agents contemplated to be of use,include, e.g., adriamycin, bleomycin, 5-fluorouracil (5-FU), etoposide(VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP),podophyllotoxin, verapamil, and even hydrogen peroxide. The inventionalso encompasses the use of a combination of one or more DNA damagingagents, whether radiation-based or actual compounds, such as the use ofX-rays with cisplatin or the use of cisplatin with etoposide.

In treating pre-cancer or cancer according to the invention, one wouldcontact the cells of a precancerous lesion or tumor cells with an agentin addition to the oncolytic adenovirus. This may be achieved byirradiating the localized tumor site with radiation such as X-rays,Wv-light, γ-rays or even microwaves. Alternatively, the cells may becontacted with the agent by administering to the subject atherapeutically effective amount of a pharmaceutical compositioncomprising a compound such as, adriamycin, bleomycin, 5-fluorouracil,etoposide, camptothecin, actinomycin-D, mitomycin C, podophyllotoxin,verapamil, or more preferably, cisplatin. The agent may be prepared andused as a combined therapeutic composition, or kit, by combining it withan oncolytic adenovirus.

Agents that directly cross-link nucleic acids, specifically DNA, areenvisaged to facilitate DNA damage leading to a synergistic,anti-neoplastic combination with an oncolytic adenovirus. Cisplatinumagents such as cisplatin, and other DNA alkylating agents may be used.Cisplatin has been widely used to treat cancer, with efficacious dosesused in clinical applications of 20 mg/m² for 5 days every three weeksfor a total of three courses. Cisplatin is not absorbed orally and musttherefore be delivered via injection intravenously, subcutaneously,intratumorally or intraperitoneally. Bleomycin and mitomycin C are otheranticancer agents that are administered by injection intravenously,subcutaneously, intratumorally or intraperitoneally. A typical dose ofbleomycin is 10 mg/m², while such a dose for mitomycin C is 20 mg/m².

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused or as alternative 5-FC may be administered and converted in atarget tissue or target cell.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageDNA, on the precursors of DNA, the replication and repair of DNA, andthe assembly and maintenance of chromosomes. Dosage ranges for X-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

Immunotherapy may be used as part of a combined therapy, in conjunctionwith mutant oncolytic virus-mediated therapy. The general approach forcombined therapy is discussed below. Generally, the tumor cell must bearsome marker that is amenable to targeting, i.e., is not present on themajority of other cells. Many tumor markers exist and any of these maybe suitable for targeting in the context of the present invention.Common tumor markers include carcinoembryonic antigen, prostate specificantigen, urinary tumor associated antigen, fetal antigen, tyrosinase(p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP,estrogen receptor, laminin receptor, erb B and p155. Antibodies specificfor CAR, integrin or other cell surface molecules, may be used toidentify cells that the adenovirus could infect well. CAR is anadenovirus receptor protein. The penton base of adenovirus mediatesviral attachment to integrin receptors and particle internalization.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, 1980. Some variation in dosage will necessarily occurdepending on the condition of the subject being treated. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

The inventors propose that local, regional delivery of oncolyticadenovirus to patients with retinoblastoma-linked cancers, pre-cancers,or hyperproliferative conditions will be a very efficient method fordelivering a therapeutically effective gene. Similarly, the chemo- orradiotherapy may be directed to a particular, affected region of thesubjects body. Alternatively, systemic delivery of expression constructand/or the agent may be appropriate in certain circumstances, forexample, where extensive metastasis has occurred.

In addition to combining oncolytic adenovirus therapies with chemo- andradiotherapies, it also is contemplated that combination with other genetherapies will be advantageous. For example, targeting of a oncolyticadenovirus in combination with the targeting of p53 at the same time mayproduce an improved anti-cancer treatment. Any tumor-related gene ornucleic acid encoding a polypeptide conceivably can be targeted in thismanner, for example, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, p16, FHIT,WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf, erb, src,fins, jun, trk, ret, gsp, hst, bcl and abl.

It is further contemplated that the therapies described above may beimplemented in combination with all types of surgery. Approximately 60%of persons with cancer will undergo surgery of some type, which includespreventative, diagnostic or staging, curative and palliative surgery.These types of surgery may be used in conjunction with other therapies,such as oncolytic adenovirus therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and microscopically controlled surgery(Mohs surgery). It is further contemplated that the present inventionmay be used in conjunction with removal of superficial cancers,precancers, or incidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection, systemic administration, or localapplication of the area with an additional anti-cancer therapy. Suchtreatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 months. These treatments may be of varying dosages aswell. Furthermore, in treatments involving more than a single treatmenttype (i.e., construct, anticancer agent and surgery), the time betweensuch treatment types may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or about 24 hours apart;about 1, 2, 3, 4, 5, 6, or 7 days apart; about 1, 2, 3, 4, or 5 weeksapart; and about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months apart,or more.

It also should be pointed out that any of the foregoing therapies mayprove useful by themselves. In this regard, reference tochemotherapeutics and non- mutant oncolytic virus therapy in combinationalso should be read as a contemplation that these approaches may beemployed separately.

V. Screening Methods

Animal models may be used as a screen for tumor suppressive effects ofoncolytic adenoviruses. Preferably, orthotopic animal models will beused so as to closely mimic the particular disease type being studiedand to provide the most relevant results. One type of orthotopic modelinvolves the development of an animal model for the analysis ofmicroscopic residual cancer cell(s) and microscopic seeding of bodycavities.

The first step in the development of an exemplary animal model is tocreate a tissue flap in the experimental animal. The term “tissue flap”means any incision in the flesh of the animal that exposes the targettissue. It is generally preferred that an incision be made in the dorsalflank of an animal, as this represents a readily accessible site.However, it will be understood that an incision could well be made atother points on the animal, and the choice of tissue sites may bedependent upon various factors such as the particular type oftherapeutics that are being investigated.

Once a target tissue site is exposed, cancer cells, either individuallyor in tumors, are contacted with the tissue site. Cancer cellapplication may be achieved simply using any convenient applicator.Naturally, this procedure will be conducted under sterile conditions.

In a particular example, 1×10⁷ cells are inoculated into the exposedtissue flap of a nude mouse. Those of skill in the art will be able toreadily determine, for a given purpose, what the appropriate number ofcells will be. The number of cells will be dependent upon variousfactors, such as the size of the animal, the site of incision, thereplicative capacity of the tumor cells themselves, the time intendedfor tumor growth, the potential anti-tumor therapeutic to be tested, andthe like. Although establishing an optimal model system for anyparticular type of tumor may require a certain adjustment in the numberof cells administered, this in no way represents an undue amount ofexperimentation. For example, this can be accomplished by conductingpreliminary studies in which differing numbers of cells are delivered tothe animal and the cell growth is monitored following resealing of thetissue flap. Naturally, administering larger numbers of cells willresult in a larger population of residual tumor cells. Those skilled inthe area of animal testing will appreciate that such optimization isrequired.

Other orthotopic animal models are well known in the art. The skilledartisan will readily be able to adapt or modify each particular modelfor his intended purpose without undue experimentation.

A. Screening for a Defective Rb Pathway

With adenovirus Δ24 and other mutant adenovirus that are unable to bindRb, it is necessary for the Rb pathway to be defective in order for thecell to transcribe and translate viral proteins. The Rb pathway isrequired to be defective in the sense that it is not able to repress thetranscription-activating activity of E2F. E2F activates thetranscription of cellular genes and adenoviral DNA if its activity isnot repressed. Examples of ways in which E2F could escape repressioninclude, but are not limited to, Rb not being able to bind E2F (i.e.,E1A binding to Rb), overexpression of E2F, less Rb than E2F andsituations in which Rb remains phosphorylated.

B. Antibodies

Antibodies can be used to detect adenoviral proteins (e.g., E1A), Rb,and other proteins of the Rb pathway. In certain aspects of theinvention, one or more antibodies may be produced that areimmunoreactive with multiple antigens. These antibodies may be used invarious diagnostic or therapeutic applications, described herein below.

As used herein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally,IgG and/or IgM are preferred because they are the most common antibodiesin the physiological situation and because they are most easily made ina laboratory setting. Means for preparing and characterizing antibodiesare also well known in the art (See, e.g., Harlow and Lane (1988),incorporated herein by reference).

Monoclonal antibodies (MAbs) are recognized to have certain advantages(e.g., reproducibility and large-scale production). The invention thusprovides for monoclonal antibodies of the human, murine, monkey, rat,hamster, rabbit and even chicken origin. Due to the ease of preparationand ready availability of reagents, murine monoclonal antibodies may bepreferred.

However, “humanized” antibodies are also contemplated, as are chimericantibodies from mouse, rat, or other species, bearing human constantand/or variable region domains, bispecific antibodies, recombinant andengineered antibodies and fragments thereof.

The methods for generating monoclonal antibodies (MAbs) and polyclonalantibodies are well known in the art. MAbs may be readily preparedthrough use of well-known techniques, such as those exemplified in U.S.Pat. No. 4,196,265, incorporated herein by reference. It is alsocontemplated that a molecular cloning approach may be used to generatemonoclonals. Alternatively, monoclonal antibody fragments encompassed bythe present invention can be synthesized using an automated peptidesynthesizer, or by expression of full-length gene or of gene fragmentsin E. coli.

C. Antibody Conjugates

Certain embodiments of the invention provide antibodies to antigens andtranslated proteins, polypeptides and peptides that are linked to atleast one agent to form an antibody conjugate. In order to increase theefficacy of antibody molecules as diagnostic or therapeutic agents, itis conventional to link or covalently bind or complex at least onedesired molecule or moiety. A reporter molecule is defined as any moietywhich may be detected using an assay. Non-limiting examples of reportermolecules which have been conjugated to antibodies include enzymes,radiolabels, haptens, fluorescent labels, phosphorescent molecules,chemiluminescent molecules, chromophores, luminescent molecules,photoaffinity molecules, colored particles or ligands, such as biotin.

Certain examples of antibody conjugates are those conjugates in whichthe antibody is linked to a detectable label. “Detectable labels” arecompounds and/or elements that can be detected due to their specificfunctional properties, and/or chemical characteristics, the use of whichallows the antibody to which they are attached to be detected, and/orfurther quantified if desired. Another such example is the formation ofa conjugate comprising an antibody linked to a cytotoxic oranti-cellular agent, and may be termed “immunotoxins”.

1. Imaging

Antibody conjugates are generally preferred for use as diagnosticagents. Antibody diagnostics generally fall within two classes, thosefor use in in vitro diagnostics, such as in a variety of immunoassays,and/or those for use in vivo diagnostic protocols, generally known as“antibody-directed imaging.” Many appropriate imaging agents are knownin the art, as are methods for their attachment to antibodies (see, fore.g., U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, eachincorporated herein by reference). The imaging moieties used can beparamagnetic ions; radioactive isotopes; fluorochromes; NMR-detectablesubstances; X-ray imaging.

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine²¹¹, ¹⁴carbon, ⁵¹chromium,³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen,iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur, technicium^(99m) and/oryttrium⁹⁰. ¹²⁵I is often being preferred for use in certain embodiments,and technicium^(99m) and/or indium¹¹¹ are also often preferred due totheir low energy and suitability for long range detection. Radioactivelylabeled monoclonal antibodies of the present invention may be producedaccording to well-known methods in the art. For instance, monoclonalantibodies can be iodinated by contact with sodium and/or potassiumiodide and a chemical oxidizing agent such as sodium hypochlorite, or anenzymatic oxidizing agent, such as lactoperoxidase. Monoclonalantibodies according to the invention may be labeled withtechnetium^(99m) by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the antibody to this column.Alternatively, direct labeling techniques may be used, e.g., byincubating pertechnate, a reducing agent such as SNCl₂, a buffersolution such as sodium-potassium phthalate solution, and the antibody.Intermediary functional groups which are often used to bindradioisotopes which exist as metallic ions to antibody arediethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraceticacid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

Another type of antibody conjugates contemplated in the presentinvention are those intended primarily for use in vitro, where theantibody is linked to a secondary binding ligand and/or to an enzyme (anenzyme tag) that will generate a colored product upon contact with achromogenic substrate. Examples of suitable enzymes include urease,alkaline phosphatase, (horseradish) hydrogen peroxidase or glucoseoxidase.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter and Haley, 1983).In particular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens and Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used asantibody binding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein byreference). Monoclonal antibodies may also be reacted with an enzyme inthe presence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate. In U.S.Pat. No. 4,938,948, imaging of breast tumors is achieved usingmonoclonal antibodies and the detectable imaging moieties are bound tothe antibody using linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

D. Immunodetection Methods

Adenoviral gene expression in a population of cells will be determinedby western blot analysis using antibodies as probes to adenoviralproteins. The level of viral proteins detected would indicate whetherviral protein expression is occurring in the cell. Immunodetection usingmonoclonal antibodies that recognize various epitopes within the Rbprotein or another protein of the Rb pathway can be used to see if Rb ora protein in the Rb pathway has been mutated.

The present invention concerns immunodetection methods for binding,purifying, removing, quantifying and/or otherwise generally detectingbiological components such as protein(s), polypeptide(s) or peptide(s)involved in adenoviral replication or the cellular Rb or p53 pathways.Some immunodetection methods include enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA), immunoradiometric assay,fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, andWestern blot to mention a few. The steps of various usefulimmunodetection methods have been described in the scientificliterature, such as, e.g., Doolittle and Ben-Zeev, 1999; Gulbis andGaland, 1993; De Jager et al., 1993; and Nakamura et al., 1987, eachincorporated herein by reference.

In terms of antigen detection, the biological sample analyzed may be anysample that is suspected of containing an antigen, such as, for example,a tissue section or specimen, a homogenized tissue extract, a cell, anorganelle, separated and/or purified forms of any of the aboveantigen-containing compositions, or even any biological fluid that comesinto contact with the cell or tissue, including blood and/or serum,although tissue samples or extracts are preferred.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. Patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

1. ELISAs

Immunoassays, in their most simple and/or direct sense, are bindingassays. Certain preferred immunoassays are the various types of enzymelinked immunosorbent assays (ELISAs) and/or radioimmunoassays (RIA)known in the art. Immunohistochemical detection using tissue sections isalso particularly useful. However, it will be readily appreciated thatdetection is not limited to such techniques, and/or western blotting,dot blotting, FACS analyses, and/or the like may also be used.

In one exemplary ELISA, an antibody that recognizes an antigen isimmobilized onto a selected surface exhibiting protein affinity, such asa well in a polystyrene microtiter plate. Then, a test compositionsuspected of containing the antigen, such as a clinical sample, is addedto the wells. After binding and/or washing to remove non-specificallybound immune complexes, the bound antigen may be detected. Detection isgenerally achieved by the addition of another antibody that recognizesthe antigen and is linked to a detectable label. This type of ELISA is asimple “sandwich ELISA”. Detection may also be achieved by the additionof a second antibody, followed by the addition of a third antibody thathas binding affinity for the second antibody, with the third antibodybeing linked to a detectable label.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes.

2. Immunohistochemistry

Antibodies may also be used in conjunction with both fresh-frozen and/orformalin-fixed, paraffin-embedded tissue blocks prepared for study byimmunohistochemistry (IHC). The method of preparing tissue blocks fromthese particulate specimens has been successfully used in previous IHCstudies of various prognostic factors, and/or is well known to those ofskill in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred etal., 1990, all of which are incorporated herein by reference).

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” tissue at room temperature in phosphate buffered saline(PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and/or pelleting again by centrifugation;snap-freezing in −70° C. isopentane; cutting the plastic capsule and/orremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and/or cutting 25-50 serial sections.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and/or embedding the block in paraffin; and/or cutting upto 50 serial permanent sections.

E. Nucleic Acid Detection

The nucleic acid sequences disclosed herein have a variety of uses. Forexample, they have utility as probes or primers for embodimentsinvolving nucleic acid hybridization. They can be used to determinewhether viral genes are being transcribed. In certain embodiments of theinvention adenoviral genes may be transcribed in cells with a mutant Rbor p53 pathways. Nucleic acid detection may be used to determine ifthere is a mutation within the Rb gene, p53 gene or other genes encodingproteins of the Rb pathway. The DNA sequences of genes of the presentinvention may be determined by methods known in the art to identifymutations within the sequence.

1. Hybridization

The use of a probe or primer of between 13 and 100 nucleotides,preferably between 17 and 100 nucleotides in length, or in some aspectsof the invention up to 1-2 kilobases or more in length, allows theformation of a duplex molecule that is both stable and selective.Molecules having complementary sequences over contiguous stretchesgreater than 20 bases in length are generally preferred, to increasestability and/or selectivity of the hybrid molecules obtained. One willgenerally prefer to design nucleic acid molecules for hybridizationhaving one or more complementary sequences of 20 to 30 nucleotides, oreven longer where desired. Such fragments may be readily prepared, forexample, by directly synthesizing the fragment by chemical means or byintroducing selected sequences into recombinant vectors for recombinantproduction.

For certain applications, for example, site-directed mutagenesis, it isappreciated that lower stringency conditions are preferred. Under theseconditions, hybridization may occur even though the sequences of thehybridizing strands are not perfectly complementary, but are mismatchedat one or more positions. Conditions may be rendered less stringent byincreasing salt concentration and/or decreasing temperature. Forexample, a medium stringency condition could be provided by about 0.1 to0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a lowstringency condition could be provided by about 0.15 M to about 0.9 Msalt, at temperatures ranging from about 20° C. to about 55° C.Hybridization conditions can be readily manipulated depending on thedesired results.

In certain embodiments, it will be advantageous to employ nucleic acidsof defined sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidinibiotin, which are capable of being detected.

2. Amplification of Nucleic Acids

Nucleic acids used as a template for amplification may be isolated fromcells, tissues or other samples according to standard methodologies(Sambrook et al., 2001). In certain embodiments, analysis is performedon whole cell or tissue homogenates or biological fluid samples withoutsubstantial purification of the template nucleic acid. The nucleic acidmay be genomic DNA or fractionated or whole cell RNA. Where RNA is used,it may be desired to first convert the RNA to a complementary DNA.

The term “primer,” as used herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty and/or thirty base pairs in length, but longersequences can be employed. Primers may be provided in double-strandedand/or single-stranded form, although the single-stranded form ispreferred.

The amplification product may be detected or quantified. In certainapplications, the detection may be performed by visual means.Alternatively, the detection may involve indirect identification of theproduct via chemiluminescence, radioactive scintigraphy of incorporatedradiolabel or fluorescent label or even via a system using electricaland/or thermal impulse signals (Affymax technology; Bellus, 1994).

A number of template dependent processes are available to amplify theoligonucleotide sequences present in a given template sample. One of thebest known amplification methods is the polymerase chain reaction(referred to as PCRTM) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1990, each ofwhich is incorporated herein by reference in their entirety.

A reverse transcriptase PCRTM amplification procedure may be performedto quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described in Sambrook etal., 2001. Alternative methods for reverse transcription utilizethermostable DNA polymerases. These methods are described in WO90/07641. Polymerase chain reaction methodologies are well known in theart. Representative methods of RT-PCR are described in U.S. Pat. No.5,882,864.

Another method for amplification is ligase chain reaction (“LCR”),disclosed in European Application 320308, incorporated herein byreference in its entirety. U.S. Pat. No. 4,883,750 describes a methodsimilar to LCR for binding probe pairs to a target sequence. A methodbased on PCRT and oligonucleotide ligase assay (OLA), disclosed in U.S.Pat. No. 5,912,148, may also be used.

Alternative methods for amplification of target nucleic acid sequencesthat may be used in the practice of the present invention are disclosedin U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497,5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905,5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB Application2 202 328, and in PCT Application PCT/US89/01025, each of which isincorporated herein by reference in its entirety.

Qbeta Replicase, described in PCT Application PCT/US87/00880, may alsobe used as an amplification method in the present invention. In thismethod, a replicative sequence of RNA that has a region complementary tothat of a target is added to a sample in the presence of an RNApolymerase. The polymerase will copy the replicative sequence which maythen be detected.

Other nucleic acid amplification procedures include Strand DisplacementAmplification (SDA), disclosed in U.S. Pat. No. 5,916,779;transcription-based amplification systems (TAS), including nucleic acidsequence based amplification (NASBA) and 3SR (Kwoh et al., 1989;Gingeras et al., PCT Application WO 88/10315, incorporated herein byreference in their entirety). Davey et al., European Application 329 822disclose a nucleic acid amplification process involving cyclicallysynthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-strandedDNA (dsDNA), which may be used in accordance with the present invention.

Miller et al., PCT Application WO 89/06700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoterregion/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Otheramplification methods include “race” and “one-sided PCR” (Frohman, 1990;Ohara et al., 1989).

3. Detection of Nucleic Acids

Following any amplification, it may be desirable to separate theamplification product from the template and/or the excess primer. In oneembodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (Sambrook et al., 2001). Separated amplification products may becut out and eluted from the gel for further manipulation. Using lowmelting point agarose gels, the separated band may be removed by heatingthe gel, followed by extraction of the nucleic acid.

Separation of nucleic acids may also be effected by chromatographictechniques known in art. There are many kinds of chromatography whichmay be used in the practice of the present invention, includingadsorption, partition, ion-exchange, hydroxylapatite, molecular sieve,reverse-phase, column, paper, thin-layer, and gas chromatography as wellas HPLC.

In certain embodiments, the amplification products are visualized. Atypical visualization method involves staining of a gel with ethidiumbromide and visualization of bands under UV light. Alternatively, if theamplification products are integrally labeled with radio- orfluorometrically-labeled nucleotides, the separated amplificationproducts can be exposed to x-ray film or visualized under theappropriate excitatory spectra.

In particular embodiments, detection is by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art. See Sambrook etal., 2001. One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

Other methods of nucleic acid detection that may be used in the practiceof the instant invention are disclosed in U.S. Pat. Nos. 5,840,873,5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729,5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244,5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124,5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227,5,932,413 and 5,935,791, each of which is incorporated herein byreference.

4. Other Assays

Other methods for screening may be used within the scope of the presentinvention, for example, to detect mutations in genomic DNA, cDNA and/orRNA samples. Methods used to detect point mutations include denaturinggradient gel electrophoresis (“DGGE”), restriction fragment lengthpolymorphism analysis (“RFLP”), chemical or enzymatic cleavage methods,direct sequencing of target regions amplified by PCR™ (see above),single-strand conformation polymorphism analysis (“SSCP”) and othermethods well known in the art.

U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavage assaythat involves annealing single-stranded DNA or RNA test samples to anRNA probe, and subsequent treatment of the nucleic acid duplexes withRNase A. For the detection of mismatches, the single-stranded productsof the RNase A treatment, electrophoretically separated according tosize, are compared to similarly treated control duplexes. Samplescontaining smaller fragments (cleavage products) not seen in the controlduplex are scored as positive.

Alternative methods for detection of deletion, insertion or substitutionmutations that may be used in the practice of the present invention aredisclosed in U.S. Pat. Nos. 5,849,483, 5,851,770, 5,866,337, 5,925,525and 5,928,870, each of which is incorporated herein by reference in itsentirety.

5. Biopsy

A tumor may be biopsied and the above tests performed upon it todetermine whether the cells have a functional Rb pathway. An example ofa biopsy protocol is as follows. The stereotactic biopsy is the preciseintroduction of a metal probe into the brain tumor, cutting a smallpiece of the brain tumor, and removing it so that it can be examinedunder the microscope. The patient is transported to the HI or CAT scansuite, and the frame is attached to the scalp under local anesthesia.The “pins” of the frame attach to the outer table of the skull for rigidfixation (frame will not and can not move from that point forward untilcompletion of the biopsy). The scan (MRI or CT) is obtained. Theneurosurgeon examines the scan and determines the safest trajectory orpath to the target. This means avoiding critical structures. The spatialco-ordinates of the target are determined, and the optimal path iselected. The biopsy is carried out under general anesthesia. A smallincision is created over the entry point, and a small hole is drilledthrough the skull. The “dura” is perforated, and the biopsy probe isintroduced slowly to the target. The biopsy specimen is withdrawn andplaced in preservative fluid for examination under the microscope. Oftenthe pathologist is present in the biopsy suite so that a rapiddetermination of the success of the biopsy can be made.

F. Diagnostic and in vitro Uses

Any of the methods above can be used in the present invention fordiagnostic and in vitro uses. The oncolytic adenoviruses of the presentinvention may be used in diagnostic assays to detect the presence ofcells with a defective Rb and/or p53 pathway. A sample of cells could beinfected with the oncolytic adenovirus of the present invention andafter an incubation period, the number of cells exhibiting adenovirusreplication can be quantified to determine the number of neoplasticcells in the sample. This may be useful to determine if the adenoviruswould be effective in treating the tumor from a patient from which acell sample was taken. Other uses are to diagnose a neoplasm as having adefective Rb and/or p53 pathway and to evaluate tumor cell loadfollowing treatment.

Alternate diagnostic uses and variations include an adenovirus with a Rbbinding mutation in the E1A or an E1B55 kD—mutation and a reporter geneto score whether cells have been transformed by detecting reporter geneexpression. Expression of the reporter gene can be correlated with aphenotype of adenoviral replication indicating a lack of a functional Rband/or p53 pathway.

VI. Polynucleotide and Polypeptide Variants

Amino acid sequence variants of the polypeptides discussed above andthroughout this application, specifically including Ang-2, NIS and hyCD,may be substitutional, insertional or deletion variants. Deletionvariants lack one or more residues of the native protein. Insertionalmutants typically involve the addition of material at a non-terminalpoint in the polypeptide. This may include the insertion of animmunoreactive epitope or simply a single residue. Terminal additions,called fusion proteins, are discussed below.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, such as stabilityagainst proteolytic cleavage, without the loss of other functions orproperties. Substitutions of this kind preferably are conservative, thatis, one amino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and include, forexample, the changes of: alanine to serine; arginine to lysine;asparagine to glutamine or histidine; aspartate to glutamate; cysteineto serine; glutamine to asparagine; glutamate to aspartate; glycine toproline; histidine to asparagine or glutamine; isoleucine to leucine orvaline; leucine to valine or isoleucine; lysine to arginine; methionineto leucine or isoleucine; phenylalanine to tyrosine, leucine ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. Accordingly, sequences thathave between about 70% and about 80%; or more preferably, between about81% and about 90%; or even more preferably, between about 91% and about99%; of amino acids that are identical or functionally equivalent to theamino acids of a particular polypeptide, such as E1A, provided thebiological activity of the protein is maintained.

The term “finctionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine, and also refers to codons that encode biologicallyequivalent amino acids.

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues or nucleotides, such as additional N- orC-terminal amino acids or 5′ or 3′ nucleic acid sequences. The additionof terminal sequences particularly applies to nucleic acid sequencesthat may, for example, include various non-coding sequences flankingeither of the 5′ or 3′ portions of the coding region or may includevarious internal sequences, i.e., introns, which are known to occurwithin genes.

In making amino acid changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biologic finction on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. It is understood that an amino acid can besubstituted for another having a similar hydrophilicity value and stillproduce a biologically equivalent and immunologically equivalentprotein.

As outlined above, amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take into consideration the variousforegoing characteristics are well known to those of skill in the artand include: arginine and lysine; glutamate and aspartate; serine andthreonine; glutamine and asparagine; and valine, leucine and isoleucine.

A. Polynucleotides

The present invention concerns polynucleotides that are capable ofexpressing a protein, polypeptide, or peptide discussed above, such asone derived from the Ang1 gene, Ang-2 gene, NIS gene or a yCD gene. Anucleic acid segment or polynucleotide encoding a Ang1 gene, Ang-2 gene,NIS or yCD polypeptide refers to a nucleic acid segment orpolynucleotide comprising a polynucleotide encoding wild-type,polymorphic, or mutant Ang1 gene, Ang-2 gene, NIS or yCD polypeptide.Included within the term nucleic acid are a polynucleotide orpolynucleotides, DNA segments, and recombinant vectors. Recombinantvectors may include plasmids, cosmids, phage, viruses, and the like. Incertain embodiments recombinant adenoviruses are contemplated. Inparticular, an adenovirus comprising an expression cassette encoding aAng1 gene, Ang-2 gene, NIS or a yCD.

Mutation may be a substitution, insertion, or deletion. In someembodiments, a mutation introduces a stop codon or introduces a frameshift that results in a premature stop codon. It is further contemplatedthat nonfunctional polypeptides may be encoded by polynucleotides, suchas truncated polypeptides. Moreover, it is contemplated thepolynucleotides of the invention may be mutated to produce a polypeptidethat lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500 ormore contiguous amino acids, including the full-length polypeptide,including the polypeptides of SEQ ID NO:2 and SEQ ID NO:4.

As used in this application, the term “polynucleotide” refers to anucleic acid molecule of greater than 3 nucleotides. Therefore, a“polynucleotide encoding a Ang1 gene, Ang-2 gene, NIS or yCD” refers toa DNA segment that contains a wild-type, a polymorphic, or a mutant Ang1gene, Ang-2 gene, NIS or yCD polypeptide; similarly, a “polynucleotideencoding wild-type Ang1 gene, Ang-2 gene, NIS or yCD” refers to a DNAsegment that contains wild-type Ang1 gene, Ang-2 gene, NIS or yCDpolypeptide coding nucleic acid or DNA sequences.

Similarly, a polynucleotide comprising an isolated or purifiedwild-type, polymorphic, or mutant nucleic acid encoding a Ang1 gene,Ang-2 gene, NIS or a yCD polypeptide refers to a nucleic acid segmentcomprising wild-type, polymorphic, or mutant Ang1 gene, Ang-2 gene, NISor yCD polypeptide coding sequences and, in certain aspects, regulatorysequences, isolated substantially away from other naturally occurringgenes or protein encoding sequences. In this respect, the term “gene” isused for simplicity to refer to a functional protein, polypeptide, orpeptide-encoding unit. As will be understood by those in the art, thisfunctional term includes genomic sequences, cDNA sequences, and smallerengineered nucleic acid segments that express, or may be adapted toexpress, proteins, polypeptides, domains, peptides, fusion proteins, andmutants. The nucleic acid encoding Ang1 gene, Ang-2 gene, NIS or yCD maycontain a contiguous nucleic acid Ang1 gene, Ang-2 gene, NIS or yCDsequence encoding all or a portion of Ang1 gene, Ang-2 gene, NIS or yCDof the following lengths: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520,530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660,670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800,810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940,950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070,1080, 1090, 1095, 1100, or more nucleotides, nucleosides, or base pairs.

“Isolated substantially away from other coding sequences” means that thenculeic acid of interest, for example the polynucleotide encoding awild-type, a polymorphic, or a mutant Ang1 gene, Ang-2 gene, NIS or yCDpolypeptide, forms part of the coding region of the nucleic acidsegment, and that the nucleic acid segment does not contain largeportions of naturally-occurring coding DNA. Of course, this refers tothe nucleic acid segment as originally isolated, and does not excludenucleic acid sequence, polynucleotide or coding regions later added tothe segment by human manipulation.

The nucleic acid segments used in the present invention, regardless ofthe length of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol.

The DNA segments used in the present invention may encompassbiologically functional equivalent Ang1 gene, Ang-2 gene, NIS or hyCDand derivative peptides. Such sequences may arise as a consequence ofcodon redundancy and functional equivalency that are known to occurnaturally within nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by human may be introduced through the application ofsite-directed mutagenesis techniques, e.g., to decrease the antigenicityof the protein or to inhibit binding to a given protein.

B. Cloning, Nucleic Acid Transfer, and Expression

Adenoviruses of the present invention can be constructed using methodsknown in the art and described herein. Expression requires thatappropriate signals be provided which include various regulatoryelements, such as enhancers/promoters that may be derived from bothviral and mammalian sources that drive host cell expression of the genesof interest. Elements designed to optimize messenger RNA stability andtranslatability in host cells also are defined.

1. Regulatory Elements

In preferred embodiments, the nucleic acid encoding a transgene productis under transcriptional control of a promoter. A “promoter” refers to aDNA sequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. The phrase “under transcriptional control”means that the promoter is in the correct location and orientation inrelation to the nucleic acid to control RNA polymerase initiation andexpression of the gene. In certain aspects of the invention a promoteris a heterologous promoter, that is the promoter is associated with anucleic acid that is not associated with its natural location.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription.

The particular promoter employed to control the expression of a nucleicacid sequence of interest is not believed to be important, so long as itis capable of directing the expression of the nucleic acid in thetargeted cell. Thus, where a human cell is targeted, it is preferable toposition the nucleic acid coding region adjacent to and under thecontrol of a promoter that is capable of being expressed in a humancell. Generally speaking, such a promoter might include either a humanor viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, rat insulin promoter and glyceraldehyde-3-phosphatedehydrogenase can be used to obtain high-level expression of the codingsequence of interest. The use of other viral or mammalian cellular orbacterial phage promoters which are well-known in the art to achieveexpression of a coding sequence of interest is contemplated as well,provided that the levels of expression are sufficient for a givenpurpose.

By employing a promoter with well-known properties, the level andpattern of expression of the protein of interest following transfectionor transformation can be optimized. Further, selection of a promoterthat is regulated in response to specific physiologic signals can permitinducible expression of a polynucleotide of interest, which may or maynot encode a polypeptide of interest. Promoters that permit expressionof a protein of interest generally under most conditions and in mostcell types is termed constitutive, and an example of this is the CMVpromoter. A tissue-specific promoter is a regulatable promoter that isallows expression only in particular tissues or cells. Tables 2 listseveral elements/promoters that may be employed, in the context of thepresent invention, to regulate the expression of the nucleic acid ofinterest. This list is not intended to be exhaustive of all the possibleelements involved in the promotion of polynucleotide expression but,merely, to be exemplary thereof.

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins. Enhancer/Promoters inlcude but are not limitedto enhancers/promoters of Immunoglobulin Heavy Chain, ImmunoglobulinLight Chain, T-Cell Receptor, HLA DQ α and DQ β, β-Interferon,Interleukin-2, Interleukin-2 Receptor, MHC Class II 5, MHC Class IIHLA-DRα, β-Actin, Muscle Creatine Kinase, Prealbumin (Transthyretin),Elastase I, Metallothionein, Collagenase, Albumin Gene, α-Fetoprotein,γ-Globin, β-Globin, c-fos, c-HA-ras, Insulin, Neural Cell AdhesionMolecule (NCAM), α1-Antitrypsin, H2B (TH2B) Histone, Mouse or Type ICollagen, Glucose-Regulated Proteins (GRP94 and GRP78), Rat GrowthHormone, Human Serum Amyloid A (SAA), Troponin I (TN I),Platelet-Derived Growth Factor, Duchenne Muscular Dystrophy, SV40,Polyoma, Retroviruses, Papilloma Virus, Hepatitis B Virus, HumanImmunodeficiency Virus, Cytomegalovirus, or Gibbon Ape Leukemia Virus.Element with related inducer inlcude MT II/Phorbol Ester (TPA) and Heavymetals; MMTV (mouse mammary tumor virus)/Glucocorticoids;β-Interferon/poly(rI)X and poly(rc); Adenovirus 5 E2/E1A; c-jun/PhorbolEster (TPA), H₂O₂; Collagenase/Phorbol Ester (TPA); Stromelysin/PhorbolEster (TPA), IL-1; SV40/Phorbol Ester (TPA); Murine MX Gene/Interferon,Newcastle Disease Virus; GRP78 Gene/A23187; α-2-Macroglobulin/IL-6;Vimentin/Serum; MHC Class I Gene H-2 kB/Interferon; HSP70/Ela, SV40Large T Antigen; Proliferin/Phorbol Ester-TPA; Tumor NecrosisFactor/PMA;Thyroid Stimulating Hormone α Gene/Thyroid Hormone; Insulin EBox/Glucose

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization.

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the nucleicacid transcript. The nature of the polyadenylation signal is notbelieved to be crucial to the successful practice of the invention, andany such sequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

C. Selectable Markers

The markers listed below can be inserted as a heterologous sequence inthe adenovirus genome. In certain embodiments of the invention, thecells contain nucleic acid construct of the present invention, a cellmay be identified in vitro or in vivo by including a marker in theexpression vector. Such markers would confer an identifiable change tothe cell permitting easy identification of cells containing theexpression vector. Usually the inclusion of a drug selection marker aidsin cloning and in the selection of transformants, for example, genesthat confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT,zeocin and histidinol are useful selectable markers. Alternatively,enzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be employed. Immunologicmarkers also may be employed. The selectable marker employed is notbelieved to be important, so long as it is capable of being expressedsimultaneously with the nucleic acid encoding a polypeptide of interest.Further examples of selectable markers are well known to one of skill inthe art.

D. Multigene Constructs and IRES

In certain embodiments of the invention, the use of internal ribosomebinding sites (IRES) elements are used to create multigene, orpolycistronic, messages. WES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message. An example ofsuch a construct is described in U.S. Pat. No. 5,665,567, which isherein incorporated by reference.

Any heterologous open reading frame can be linked to IRES elements. Thisincludes genes for secreted proteins, multi-subunit proteins, encoded byindependent genes, intracellular or membrane-bound proteins andselectable markers. In this way, expression of several proteins can besimultaneously engineered into a cell with a single vector and a singleselectable marker.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those skilled in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Delta 24 Studies

A. Material and Methods

Regional Glioma Models. U87MG and U251MG glioma cell human xenograftmodels utilize human tumor cell lines obtained from patients, which areplaced into short or long-term tissue culture. The advantages of thissystem are that the tumor cells are of human origin and have readilyidentifiable characteristics such as expression levels of various growthfactors and receptors. These studies, however, require immunocompromisedanimals to prevent tumor rejection, eliminating analysis of the role ofthe immune system in tumor biology and response to treatments. Mostxenograft models are well established and form predictable and reliableintracranial tumors with fairly uniform animal deaths occurring at 20-22days. However, these tumors lack the heterogeneity seen in the clinicalsetting. Furthermore, most xenograft models do not demonstrate extensiveinvasion of the surrounding brain parenchyma. The implanted tumors tendto grow spherically and animal death is related to intracranial pressureassociated with ventricular system compression and herniation, as seenafter necropsy. For assessing antiangiogenic treatments, particularlywith a non-invasive imaging component, the localized and sphericalgrowth of the xenograft implant may be an advantage and has been usedextensively for this purpose. The defined localization of the tumorsimplifies the correlation of histopathologic findings with imagingresults (FIG. 4).

U87MG human xenograft model implanted intracranially into the mouseputamen has a demonstrated reliability (nearly 100% tumor production)and highly predictable growth and survival parameters (median survival21 days rt 1 day). Furthermore, use of the intracranial screw technique(Lal et al., 2000) ensures reproducible placement of tumor and theability to access tumor either for sampling or for injecting treatments(local delivery of viruses) or markers into the tumor mass. Anadditional feature that improves the reproducibility of this techniqueis the use of a pump that simultaneously injects prepared tumor cellsinto 10 animals. This technique produces uniform injection times andconsistent quality of the cell preparations.

Systemic Lung Cancer Models. Reproducible lung cancer models areroutinely used to take advantage of distinctly different patterns oforgan involvement. Lung cell lines H1299 and Δ549 when injected throughtail veins, develop very reproducible systemic animal tumor burdens. Inthe case of H1299, strictly bilateral lung tumors are produced. Incontrast, when an A549 cell line is used, all systemic organs (exceptthe CNS) are at risk for tumor development.

Retinoblastoma Pathway Status. The Rb pathway status has been evaluatedin various cell lines, including the cell lines described herein:(U87MG, U251MG, H1299, and A549). The Saos-2 cell line is used for theRb -null and Rb restoration studies.

Coxsackie Adenoviral Receptor (CAR) in the Cell Lines Used. To ensurethat the cell line can be infected by adenovirus, including Δ24 andΔ24-NIS the status of CAR expression was assessed in the various celllines.

Imaging Models. Nuclear imaging may detect radiation signals fromradiopharmaceuticals that have been systemically administered toexperimental animals. Conventional nuclear imaging is performed using agamma camera that can detect photons with energies between 60 and 600 eV(usually between 80 and 400 keV). One of the advantages to whole bodyscanning is rapid sequential imaging (typically 1 image per 30-60seconds) and the ease by which animals can be anesthetized and placed ina lane-prone position. The utility of this system is also evident by itswidespread use in clinical medicine, thus creating a convenient bridgebetween animal and human studies.

B. Initial Results

Δ24 Oncolytic Adenovirus. The conditionally replication selectiveadenovirus Δ24, has been shown to selectively replicate in tumor cellsthat have a functional defect in the p16/Rb/E2F pathway, a defect thatoccurs in more than 90% of malignant glioma tumors. To determine if Δ24replicates in vivo, studies were conducted in which human U87MG gliomacells are injected (5×10⁵) intracranially in nude mice. Three dayslater, 1×10⁸ plaque forming units of Δ24 are injected into the tumorusing a screw-guided system. The animals are sacrificed arbitrarily onday 25 post-treatment to examine the extent of Δ24 adenoviraldissemination within the tumor.

Microscopic examination shows a highly vascularized glioma localized inthe right frontal basal ganglia. Three different areas are observed: (1)central area of necrosis, (2) highly infected glioma cells(characterized by nuclear viral inclusions) and, (3) minimallyinfected/uninfected tumor tissue. Previous studies showed that a 25-dayU87MG xenograft treated with Δ24 does not demonstrate areas of necrosisgreater than 10% of the total volume of the tumor. In tumor-bearinganimals treated with Δ24, the areas of necrosis (debris from viralinfected lysed tumor cells) are approximately equivalent to 30% -60% ofthe total volume of the tumor. The infected cells, are easilyidentifiable because they display viral nuclear inclusions, surround thearea of necrosis, and are separated from normal brain tissue by a zoneof minimally infected versus uninfected cells (FIG. 5). This pattern ofclearly different areas suggests that the virus spreads from the centerof the tumor to its periphery.

The tumors are examined after immunostaining with an antibody specificfor the adenovirus hexon protein (an encoded adenoviral late gene). Thepresence of hexon protein indicates and correlates well with viralreplication. The characteristics of the staining in both nuclear andcytoplasmic regions, are due to the presence of capsid proteins producedin the cytoplasm and transported to the nucleus for viral assembly.

Transgene Expression by Δ24 Virus. The expression of an exogenoustransgene mediated via Δ24 transport (as an effective delivery vector),is described herein. Others have shown exogenous transgene expressionwith the use of other oncolytic viral vectors. Based on western blotanalysis and quantitative RT-PCR analyses (FIG. 6), which demonstratedthat a tumor-selective oncolytic adenovirus (Δ24-hyCD) can be used toefficiently transduce high levels of an exogenous hyCD gene, resultingin a potent anti-glioma effect in vitro and in vivo. This finding isimportant because gene therapy has been hampered by various obstacles(Roth and Cristiano, 1997). In particular, gene therapy is hampered byinadequate nucleic acid delivery to a large number of tumor cells.Targeting of tumor cells and extension of lethality to tumor cells thatare a modest distance away from the major tumor mass by a “bystandereffect” (Springer et al., 2000) is one aspect of this invention. Viralconstructs were designed to gain tumor selectivity by targeting thedefective p I6/Rb/E2F pathway in malignant gliomas by using Δ24adenovirus that contains a deletion in the E1A gene. This technique isdesigned to improve exogenous nucleic acid delivery to tumor cells byusing a replication-competent adenovirus and to obtain a bystandereffect via transgene expression.

Tumors such as malignant gliomas, which are intrinsically resistant toradiation therapy and chemotherapy and recur exclusively in a localmanner, are excellent candidates for gene therapy strategies(Puumalainen et al., 1998). FIG. 6 shows that an exogenous nucleic acidproduct (hyCD encoding polynucleotide) can be effectively expressed intarget tumor cells by using the Δ24-hyCD oncolytic virus. The hyCDencoding polynucleotide is expressed at very high levels and is able toactively convert 5-FC into 5-FU, which exhibits superior activityagainst glioma cell lines. Notably, adding 5-FC to cell cultures oradministering it to animals treated with Δ24-hyCD did not interfere withthe oncolytic potential of the virus. One concern is that the hyCDencoding polynucleotide will not have enough time to be efficientlyexpressed in an oncolytic setting. The inventors are also concerned thatadding 5-FC could “poison” the infected producer cells, thereby bluntingthe oncolytic effect. The inventors selected a gene dependentenzyme/pro-drug therapy (GDEPT) strategy using the Δ24-hyCD oncolyticvirus for additional study. The results show that the system provides awindow of opportunity that is sufficient to produce a dramaticexpression of exogenous nucleic acid without abrogating effectiveoncolysis. It was also found that the optimal production of hyCD mRNA istime- and dose-dependent with respect to the particular cell lineinfected. Specifically, U251 MG cells can produce approximately a 3-loghigher amounts of mRNA than U87MG cells (FIG. 7). This finding mayrelate to differences in the rate of infectivity of U87MG versus U251MGcells (Suzuki et al., 2001). It was also found that input titer andincubation time strongly affected hyCD mRNA production in a cellline-specific fashion. These results were confirmed by pathologicexaminations confirming the desired effect. Histopathologic sections ofbrains from animals treated with Δ24-hyCD recapitulate thecharacteristic process of “zonal” replicative advancement of aninfectious “wave” propagating through a tumor mass. Moreover, theexogenous genetic burden of hyCD, with or without 5- FC, did not seem toimpede this infectious wave within the tumor. The realization thatnucleic acid delivery strategies can be effectively accomplished when anoncolytic or replication-competent virus is used makes devising improvedtherapeutic strategies plausible (Hermiston et al., 2000). Severalreports have documented improved infectivity by inserting an RGR motifinto the Δ24 fiber-knob (Suzuki et al., 2001); improved selectivity inan environment in which cells are actively dividing; and, recently, animproved anticancer effect accomplished by combining radiotherapy withoncolysis.

Autoradiography. Whole-mount sections of animals were prepared aftertail vein injection of ¹⁸⁸Re elutant produced by a Rhenium generator at100 μCi, which provides additional conformation of the anatomiclocalization of the images provided by gamma camera assessment. Besidesthyroid tissue, NIS expression is present in the gut (predominantly thestomach) as can be demonstrated in the images shown in FIG. 8. Thebladder where the isotope is being eliminated is also visualized.Individual organs can be harvested and isotope counts can be determinedper gram of tissue as a quantitative measure of radionuclide uptake. Asshown in FIG. 9 uptake of ¹⁸⁸ReO₄.

Sodium/Iodide Symporter In Vitro Activity. U251MG and U87MG glioma celllines are transiently transfected, then incubated with ^(99m)TcO₄. Thesein vitro techniques are modifications of an assay developed by Petrichet al. (2002). Briefly, cells are plated into 24-well plates and allowedto achieve 90% confluence. Various titers of Δ24-NIS are added,incubating and infecting the cells for 2 h. The cells are then washed,and the assay performed at 24, 48, and 72 h. The cells are pre-incubatedat 37° C. for 30 min in 1 ml of HEPES-buffered Hanks balanced saltsolution (bHBSS). 18.5 to 37.0 kBq per ml(0.5-1 mCi per ml) of^(99m)-TcO₄ will be added after a 1 h incubation time. The medium isremoved and the cells washed twice with ice-cold bHBSS. Cellularradioactivity is released with 1 ml ice-cold 100% ethanol for 20 min,and counted in a cross-calibrated gamma counter. Protein content of the6 wells per cell line are determined using a BCA assay kit.

Cell Mixing Experiments. Because of the concern that an oncolytic viruscould destroy the cells expressing the NIS transgene and blunt theability to concentrate-adequate amounts of radionuclide, a study wereperformed to determine the percentage of cells required to obtainsignificant concetrations of isotope. These cell-mixing experiments wereperformed in a similar fashion to the ^(99m)TcO₄ accumulation studiesfor stable clone expression studies shown in FIG. 10. The mixing studieswere performed by first infecting U251 cells with Δ24-NIS and then afterwashing, mixing various concentraions of infected cells with uninfectedU251 cells. The result is shown in FIG. 11 and is expressed as foldincrease (ratios) over control cells. These studies demonstrate thateven at 10%, cells expressing NIS from Δ24-NIS accumulate approximately10-fold Tc^(99m)O₄ over control cells. This also demonstrates theability of adenovirus to express transgenes to much higher levels thaneven stable transfected cell lines.

En-Bloc Tumor resection. Adenoviral gene therapy trials have beenrecently completed, adenoviral P53 (Ad-P53) phase I clinical trial.Patients with recurrent malignant glioma were enrolled after meetingentry criteria and underwent stereotactic biopsy with subsequentinjection of Ad-p53. The injection catheter was cut at the level of thedura and ligated to maintain a geographic marker of the actual injectionsite. Two weeks after injection the patient returned for en bloc tumorresection under the direction of Dr. Frederick Lang, M.D. AndersonCancer Center. The tumor mass was carefully dissected circumferentially,preserving the architecture along with the injection catheter andinternal injection site. The en bloc tumor specimen was sectionedperpendicular to the catheter placement to allow inspection andmeasurement of the distance of viral spread. As demonstrated in FIG. 12,this technique allows one to correlate the distance of oncolytic viralspread with data obtained from the Ad-P53 clinical trial, as well ascorrelating the results with imaging findings obtained prior to tumorresection.

FIG. 12 shows a specimen from a patient treated with a single injectionof Ad-P53 at a dose of 3×10¹⁰ vp in 1 ml. FIG. 12A is a photograph of asurgical specimen that was removed en bloc. The injection catheter isprotruding from the tumor. FIG. 12B are formalin-fixed tumor blocks.Specimen shown in panel a has been cut to the catheter. The hole createdby the catherter is evident. FIG. 12C is a low-power view (300×) of thesame immnunostained with antibody to p53 protein. The hole from thecatheter is at the top of the photograph. Transfected tumor cells staindarkly and are distributed within 5 mm of the injection site. FIG. 12Dis a view (500×) of the same section as FIG. 12C demonstrating positiveimmunostaining for P53 within the transfected cells. FIG. 12E is a viewof adjacent section of that shown in FIG. 12 C demonstrating stainingfor p21/waf in same distribution as P53 staining. FIG. 12F shows a lowpower (10×) view of cross-section. The catheter was within the centralhole. Blue staining around hole shows distribution of Ad-P53.

Example 2 Construction and Characterization of Delta 24-NIS

To determine whether the expression pattern of NIS is controlled by thestatus of the Rb pathway in Δ24-NIS-infected glioma cells the inventorsstudied the correlation between an E2F-promoter driven NIS expressionwith the replication ability of Δ24 in glioma cells and normal humanastrocytes with different cell cycle profiles (from quiescence to activeproliferation). The expression of NIS under the control of thetumor-specific promoter hTERT was also studied. Furthermore, thecapability of Δ24-NIS infected cells to effectively take up variousradionuclides was characterized.

A. Material and Methods

Materials and Methods used in these studies include the construction andcharacterization of a replication-competent Δ24 adenovirus encoding atherapeutic or diagnostic transgene, e.g., Δ24-hyCD or Δ24-NIS, seeschematic representation shown in FIG. 13. Also described are thematerial and methods used for assessing transgene expression in varioustumor types and under the controls of diverse promoters to demonstrateits applicability to multiple, varied tumors. Exemplary materials andmethods include”

NIS in vitro activity. Cloning of the human NIS into pcDNA3.1-Zeo(invitrogen) will be preformed by RT-PCR of human thyroid mRNA. U251 MGand U87 MG glioma cell lines will be transiently transfected, thenincubated with ^(99m)TcO₄. These in vitro techniques have already beencarried out in our laboratory and modified based on an assay developedby Petrich et al. Briefly, cells will be plated into 24 well plates andallowed to achieve 90% confluence. Various titers of Δ24-NIS will beadded, incubating and infecting the cells for 2 hrs. The cells will thenbe washed, and the assay performed at 24, 48, and 72 hrs. the cells willbe pre-incubated at 37° C. for 30 min in 1 ml of HEPES-buffered Hanksbalanced salt solution. 18.5 to 37 kBq per ml (0.5-mCi per ml) of^(99m)TcO₄ will be added after a 1 hr incubation time. The medium willbe removed and the cells washed twice with ice-cold bHSS. Cellularradioactivity will be released with 1 ml ice-cold 100% ethanol for 20min, and counted in a cross-calibrated γ counter. Protein content of the6 wells per cell line will be determined using a BCA assay kit.

Because of the concern that an oncolytic virus could destroy the cellsexpressing the NIS transgene and blunt the activity to concentrateadequate amounts of radionuclide, studies were performed to determinethe percentage of cells required to obtain significant concentrations ofisotope. These cell mixing studies were performed in a similar fashionto the ^(99m)TcO₄ accumulation experiments for the stable cloneexpression experiments shown in FIG. 10 the mixing experiments wereperformed by first infection U251 cells with Δ24-NIS and then afterwashing, mixing various concentrations of infected cells with uninfectedU251 cells with Δ24-NIS and then after washing, mixing variousconcentration of infected cells with uninfected U251 cells. The resultis shown in FIG. 11 and is expressed as fold increase (ratios) overcontrol cells. These experiments demonstrate that even at 10% cellsexpressing NIS form Δ24-NIS accumulate approximately 10 fold Tc⁹⁹O₄ overcontrol cells. This also demonstrates the ability of adenovirus toexpress transgenes to a much higher levels than even stable transfectedcell lines.

E2F or hTERT Driven Transgene. Exemplary E2F-driven NIS expressionvectors are constructed in a fashion similar to the construction ofcytomegalovirus (CMV) driven NIS. Specifically, the CMV promoter onpcDNA3.1-Zeo (Invitrogen) is excised using restriction enzymes Apa I andNhe I, and the promoters for E2F and hTERT are amplified by PCR withprimers that contain these restriction sites at the 5′-ends of theirsequence. These promoters are cloned into the immediate proximal regionof the NIS gene. Replication competent viruses are produced by cloninginto the appropriate shuttle vectors. These shuttle vectors are thenco-transfected into 293 cells for recombination to active virus. Theseviral constructs are used in subsequent studies to determine theirefficiency of expression against various cellular backgrounds.

Different promotor constructs are produced by using standard cloningtechniques. For example hTERT-NIS (plasmid p-hTERT was kindly providedby Dr. Fang, M.D. Anderson Cancer Center) and have the NIS encodingpolynucleotide inserted between the Xhol and HindIII sites just proximalto the NIS encoding polynucleotide. This cassette is amplified by PCRand cloned into a shuttle vector. The methods culminating withco-transfections into 293 cells.

Similarly, the E2F promoter will be obtained from plasmid pE2F1-neo(kindly provided by Dr. Ta Jen Liu, M.D. Anderson Cancer Center):however, both the promoter and final cassettes will typically be clonedby PCR amplification. The steps described above are repeated to obtainΔ24 containing NIS or hyCD under the control of E2F or hTERT.

Cell lines and culture conditions. U87MG cells, A549 cells, and H1299cells (obtained from the American Type Culture Collection, Manassas,Va.), U251 MG human glioma cell lines (kindly provided by Dr. Yunglaboratory), and Saos-2 cell line (kindly provided by Dr. Fueyo'slaboratory) are cultured in Dulbecco's modified Eagle/F12 medium (1:1,vol:vol) (Media Tech, Hermdon, Va.) containing 5% fetal bovine serum(DIFCO) and 2 nM glutamine. Cells are grown in culture at 37° C. at 5%CO₂ without antibiotics and passaged fewer than 12 times during thestudies.

Adenoviruses. Construction of the Δ24 adenovirus has been describedelsewhere (Fueyo et al., 2000, incorporated herein by reference). Thisconstruct has a 24 bp deletion in the CR2-region of the E1A gene(nucleotides 923 to 946, both included) corresponding to the amino acidsL₁₂₂TCHEAGF₁₂₉.

To construct Δ24-NIS oligonucleotides spanning the full length of NISare used to obtain the first clone of the human homologue of thetransport pump. Oligonucleotide primers (Midland Certified Reagent Co.,Midland, Tex.) with the following sequences are used: Forward primer:5′-AGCCTGTGCAATCAGGGTC-3′ (SEQ ID NO:7) and Reverse primer:5′GGGTACCATATGCGCT-3′ (SEQ ID NO:8).

After a full-length (1.9 kb) fragment is obtained, it is subsequentlycloned into the mammalian expression vector pcDNA3.1-Zeocin(Invitrogen). In addition, the 5′-distal region contains the naturalKozac sequence. Colonies are isolated after transformation into DH5-αcompetent cells. Bacterial clones are picked and their plasmidssequenced. Several clones with the DNA sequence of interest are thentransiently and stably transfected into U87MG and U251MG glioma celllines and assayed for enzyme activity. Suitable clones expressing enzymeactivity are cloned into the E3 region of pBHG10 (Microbix). pBHG10-hNISand pXC1-Δ24 are cotransfected into 293 cells to allow homologousrecombination, as previously described (Fueyo et al., 2000).

The viruses are propagated in 293 cells and purified byultracentrifuigation in a cesium chloride gradient. All viruses aretitered using a plaque method as well as optical density measurements.Viruses are maintained at −80° C. until used. Single lots of adenovirusΔ24, adenovirus Δ24-hyCD and adenovirus Δ24-NIS are used in thefollowing studies. As controls, non-replication-competent Ad-5 is usedas a control (E1A-deleted with the NIS encoding polynucleotide clonedinto the E1A region). Additional controls of wild-type adenovirus aswell as Δ24-NIS that had been inactivated by UV light and cells that hadbeen mock-infected with culture medium are also used.

In vitro expression of NIS. In vitro expression studies are todemonstrate that the use of NIS in a Δ24 vector can concentrate variousradioisotopes, including ^(99m)TcO₄ and ¹²³I or ¹³¹I, within anoncolytically infected xenograft glioma tumor. These studies are carriedout in tissue culture using the U251MG, U87MG and D54 glioma cell lines.These in vitro techniques have been modified by the inventors based onan assay developed by Petrich et al. Briefly, cells are plated onto24-well plates and allowed to achieve 90% confluence. Various titers ofΔ24-NIS are added and allowed to incubate and infect the cells for 2 h.Cells are prepared and assayed at 24, 48, and 72 h. The cells arepre-incubated at 37° C. for 30 min in 1 ml of HEPES-bufferedHanks-balanced salt solution (bHBSS). 18.5 to 37.0 kBq per ml (0.5 to 1mCi per ml) of ^(99m)TcO₄ is added after 1 h of incubation time. Themedium is removed and the cells are washed twice with ice-cold bHBSS.Cellular radioactivity is released with 1 ml ice-cold 100% ethanol for20 min, and counted in a cross-calibrated gamma counter. Protein contentof the 6 wells per cell line will be determined using a BCA assay kit.Sodium perchlorate (NaClO₄) is used to inhibit the NIS pump and as anegative control.

Real-time Quantitative Polymerase Chemical Reaction (PCR). To assesstransgene expression in tumor cell lines infected with Δ24-NIS,quantitative RT-PCR is used. U251MG and U87MG cell lines are grown to95% confluency, harvested with 0.25% trypsin/EDTA, replanted into T25flasks to a total of 2×10⁶ cells; and incubated overnight. The media isaspirated and 2 ml of adenovirus Δ24-hyCD or Δ24-NIS will be added at0.1, 1, 5, 10, or 100 MOI to duplicate samples from a viral stock of1×10¹¹ pfui/ml, and incubated for 1 h with continuous shaking. The virusis aspirated and cells are washed twice twice with phosphate-bufferedsaline (PBS). Fresh complete medium containing 10% fetal bovine serum(FBS) is replaced and the cells incubated at 37° C. for 24, 48, 72, or96 h. The cells are washed twice with PBS. Floating cells arecentrifuged, immediately frozen, and stored at −80° C. before harvestingmRNA. The cell pellets are lysed with Trizol reagent (Life Technologies)and the RNA is purified according to the manufacturer's recommendationsfor subsequent amplification by TAQ-Man. A forward primier sequence of5′-CAACATGAGGTTCCAGAAGGG-3′ (SEQ ID NO:9), a reverse primier sequence of5′-CAGTTCTCCAGGGTGGAGATCT-3′ (SEQ ID NO:10) and a TaqMan probe with aseqeunce of 5′-TCCGCCACCCTG CACGGC-3′ (SEQ ID NO:11) are used for theamplification of NIS.

The primers or probe are typically labeled with a FAM label at the 5′end and TAMRA label at the 3′ end for human NIS. Control primers andprobes are used for the S9 housekeeping gene. The expression of mRNA forNIS is quantified and reported relative to a stable NIS expression cloneof the U251 MG glioma cell line. Expression levels are determined byusing the ABI 7000 sequence detection system (Applied Biosystems, FosterCity, Calif.).

Western Blot Analysis. U251MG and U87MG cell lines are prepared in6-well plates and treated with Δ24, Δ24-NIS, nonreplication competentAd-NIS, or PBS (mock-treatment), as described above. The cells areharvested at 24, 48, 72, or 96 h after treatment. Total cell lysates areprepared by incubating cells with 1× sodium dodecyl sulfate (SDS) samplebuffer (62.5 mM Tris-HCl pH 6.8, 2% w/v SDS, 10% glycerol, 50 mMdithiolthreitol). Protein concentration are typically quantified using abicinchoninic acid (BCA) method (Pierce, Rockford, Ill.). Proteinsamples (20 μg) are boiled at 98° C. for 5 min, and lysates separated ona 15% SDS-Tris glycine polyacrylamide gel, while being subjected toelectrophoresis at 95 V for 2 h. The seperated proteins are thentransferred to a nitrocellulose membrane. The membrane is blocked with3% nonfat milk, 0.05% Tween 20, 150 mM NaCl, and 50 mM Tris (pH 7.5) andincubated with primary antibody for hNlS (provided by Brahms Institute,Germany). Typically the secondary antibody is horseradishperoxidase-conjugated goat antimouse IgG (Pierce, Rockford, Ill.). Themembranes are developed according to Amersham's enhancedchemiluminescence protocol (Amersham Corp., Arlington Heights, Ill.).

Cell Viability Assays. U251MG and U87MG cell lines are grown to 95%monolayer confluence, trypsinized and harvested with 0.25% trypsin/EDTA,plated in 6-well tissue culture plates, and allowed to adhere overnightat 37° C. in 5% CO₂ in humidified incubators. The cell lines are thentreated with various concentrations of virus, either with Δ24, Δ24-NIS,nonreplication competent Ad-NIS, UV-inactivated Δ24-NIS or PBS (MOIranging from 0.1 to 100). The addition of radionuclides at variousconcentrations will not exceed a 2 mCi total dose. The cells will beincubated at 37° C. for 5 days. Cell viability is determined by cellularrespiration using 3-(4,5-methylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium)(Promega, Madison, Wis.) as suggested by manufacturer. The cell survivalfraction is measured at each drug concentration as the ratio ofabsorbance at 490 nm. This calculation is normalized for backgroundabsorbance of the culture medium alone. The cell survival fraction isplotted against the logarithm of the drug concentration, and ICW valuesextrapolated via linear regression into the drug concentrationsproducing a 50% reduction of normalized absorbance. Crystal violetassays are performed, as described Fueyo et al. (2000), to determine theoncolytic potential of the various constructs with virus and isotopes asdescribed for MTT assays.

Tumor kill derived from oncolytic virus versus radiotherapy isdisteinguished by FACS analysis, as well as detection of apoptoticmarkers, since cell death from adenovirus is mediated through cell lysisrather than apoptosis.

RB-null and RB-restored Cell Lines. To confirm that Δ24-NIS adenovirusreplicates in a cell-cycle restricted manner when the Rb pathway isfunctionally normal, the ability of the mutant adenovirus to replicatein arrested cells expressing wild-type Rb is evaluated. For theseexperiments, Saos-2 osteosarcoma cell line is used. Saos-2 cells have awell-characterized disruption of the Rb pathway, have awell-characterized response to the transfer of exogenous Rb, and can beeasily infected with adenovirus. Saos-2 cells are infected with 100 MOIof an adenoviral vector carrying the exogenous wild-type Rb cDNA or theAd5CMV-pA adenovirus. 72 hrs later the cells are infected with 10 MOI ofeither the E1A-mutant adenovirus or the UV-Δ24-NIS adenovirus. It iscontemplated that cells pretreated with a vector control, Ad5CMV-pA willbe permissive for adenoviral replication and expression of the reportergene. By contrast, cells infected with the Ad5CMV-Rb vector shouldacquire an oncolytic-resistant phenotype with an inability to displaydisseminated NIS expression. Because the effect of the E1A-mutantadenovirus is theoretically restricted by cell-cycle factors, flowcytometric analyses of DNA content is used to monitor the changes in thecell-cycle profile of Saos-2 cells in parallel to the studies describedherein. These studies should explain if restoring Rb renders the Δ24-NISvirus unable to efficiently induce the entry of cells into the S phase,therefore precluding viral replication and a high expression of NIS. Tofurther confirm the virus-suppressive effect of the Rb protein, theeffect of cyclin-dependent-kinase inhibitor p21, a regulator of Rbfunction, will be evaluated for reducing the effect of the E1A-mutantadenovirus on the viability of wild-type Rb cells and the expression ofNIS. D54MG cells are infected with 100 MOI of an adenoviral vectorcarrying the exogenous wild-type p21 cDNA or Ad5CMV-PA, and 3 days lateris infected with the Δ24-NIS virus at 10 MOI.

Statistical analysis. Graphical displays and descriptive summarystatistics, including means (standard deviations) and medians (range)for continuous variables, are used. Ninety-five percent confidenceintervals are estimated. Categorical outcomes will be summarized usingcontingency tables.

Construction Δ24-NIS. The human form of NIS (HNIS) has been cloned andits activity demonstrated by transient transfection in glioma celllines. The Δ24-NIS adenoviral construct has recently been obtained andsuperior radionuclide accumulation with this virus has been seen ascompared to stably transfected cell lines. In other embodiments of theinvention a transgene may be put under the control of an induciblepromoter system, such as the Tet/on or Teffoff systems.

Example 3 Characterization of in vivo Effects of RadionuclideAccumulation for Imaging Tumors

These studies are conducted to determine the level of Δ24-NISpropagation in intracranial glioma tumor-bearing animals through theimaging of various radionuclides, to correlate imaging at multiple timepoints with pathologic material to determine regional aspects of viralspread throughout tumors both in intracranial gliomas and systemic lungcancer models with liposome:Δ24-NIS complexes, and to determine, by invivo imaging, the efficiency and specificity of delivery of Δ24-NISmediated mesenchymal stem cell deliverv to a systemic lung cancer model.

A. Characterizing the in vivo Effects of Radionuclide Accumulation forImaging Tumors

It is critical to the future of oncolytic gene therapy trials to havethe ability to monitor the infective spread of virus throughout a tumor.A sensitive and convenient method is needed to image this viralprogress. This may have implications in the future for administeringantiviral therapies to a patient after a tumor has been effectivelyeradicated, or perhaps in providing a patient with immunosuppressivemedications to allow the oncolytic virus to adequately destroy thetarget tumor without interference by host immune responses. Theseprocesses would be very difficult to assess without the ability to trackthe progress of the oncolytic virus through an imaging-based system. Forthis reason the development of imaging techniques using NIS will bestudied in animal models using various isotope detection techniques.Additionally, the possibility of tracking the oncolytic virus Δ24-NISboth after direct intratumoral injection (the intracranial glioma model)and following a method of systemic delivery using DOTAP: cholesterolencapsulated in Δ24-NIS (in the systemic lung cancer models) is studied.These studies will establish a basis for clinical trials for effectivelyimaging the progression of oncolytic therapies. The objective of thisstudy is to determine the level of Δ24-NIS propagation in intracranialglioma tumor-bearing animals and systemic lung cancer models throughimaging radionuclide expression using different delivery methods.

U251MG and U87MG glioma cell lines will be injected intracranially into4 to 6 week old nude mice. After 5 days of tumor growth Δ24-NIS viruswill be injected intracranially. After 5 days, the animals will beanesthetized and injected through the tail vein with a radioisotope(e.g., ^(99m)TcO₄ or ¹³¹I). The mice will then be positioned on a gammacamera with a 140 kev-high resolution colimator. The images will beobtained at 5, 15, 30, 60, 90 and 120 min after injection of theradioisotope. These time points have been selected because the optimalsignal to noise ratios (SNR) will not initially be known. Because theclearance of isotope and uptake by the NIS pump may vary in differentanimals, the maximum SNR observed for each animal (M_(obs)SNR) forcalculations at any given time point is used.

Radioiodide-uptake assay. U87MG and U251MG glioma cells are seeded in6-well mictrotiter plates (5×10⁵ cells per well) and incubated for 24 hat 37° C. with 5% CO2 and 10% FBS. On the following day, cells arewashed twice with PBS and replaced with fresh media without FBS. Thereplacement media will contain either 2.0 μCi of Na¹²³I along with 5 μMof NaI as a carrier. The cells will then incubate for 1 h and thenwashed with ice-cold Hanks-balanced salt solution (HBSS). The cells willbe lysed with 95% ethanol, counted, and measured using a gamma counter(Packard Instruments, Ill.). All studies will be performed in triplicate(Haberkorn, 2001; Cho et al., 2002).

Radioiodide (Autoradiography) assay In Vivo. U87MG and U251 MG gliomacells are harvested by trypsinization after being washed with PBS cellsand 5×10⁵ cells concentrated in 10 μl of serum-free sterile medium. Thecell suspension is injected intracranially into 4 to 6 week old nude(nu/nu) mice using a screw-guide technique. Five days postglioma-injection, the mice will be injected once with 1×10⁸ PFU Δ24-NISvirus using the screw-guide method. Five days post-viral injection, themice will have radioisotopes (e.g., I¹²³ or I¹³¹) injected through atail vein at a dose of about 0.5 μCi per gram of animal body weight. Twohours after tail-vein injection the animals are euthanized and tissuesperfused with formalin/saline for autoradiography. The brain and othertissues are harvested and sectioned for gamma-autoradiography using acounter Cobra E5003 device (Packard instruments). The radioactivity isexpressed in (counts of tissue of interest per milligcam of tissue ofinterest)/(the counts obtained in the liver per milligram of livertissue).

Imaging. Whole body gamma camera imaging will be performed in the smallanimal imaging facility at M.D. Anderson, Houston, Tex., which includesa dedicated 4.7 Tesla animal MRI, animal CT, animal Single PhotonEmitted Computed Tomography (SPECT), as well as a purchased animalPositron Emission Tomography (PET) scanner. For the animal modelsdescribed, multiple imaging techniques will be used both to gaininformation regarding the spread of Δ24-NIS and to correlate andvalidate the findings from the various imaging techniques used.Specifically, small animal MR imaging with a 4.7 Tesla Brucker magnetwill be performed in the small animal facility to ensure uniform tumortake. Additionally, whole animal gamma camera imaging will be performedwith a dedicated mouse gamma camera unit, also in the small animalimaging facility.

The Emission Tomography nuclear imaging technique measures emittedradiation signals from different locations and reconstructs the imagesbased on the geometry of detection to provide the exact locations of theinitial source's signals. As in PET imaging, this method takes advantageof compounds that emit positrons that undergo very rapid annihilationwith neighboring electrons and yields 2 gamma photons, which exit at180° from each other. More complete geometric information is obtained bythe simultaneous capture of gamma photons emitted in oppositedirections. In the proposed studies, ¹²⁴I, which has a half-life ofapproximately 4 days, is ideally suited for the evaluation of theΔ24-NIS construct. In SPECT, the measured radiation is detected fromdifferent projections, using a conventional gamma camera. This approachtypically involves a dual-head gamma camera system, which allows for thedetection and geometrical calculation of its emission source. Theadvantage of SPECT imaging is the reduced cost of using more readilyavailable gamma-emitting radioisotopes, such as ^(99m)TcO₄, ¹²³I, or¹³¹I. Another advantage of using nuclear imaging to detect oncolyticviral spread is that such small quantities of radioisotope are requiredfor adequate imaging (typically much less than microgram quantities) andonly minimal pharmacodynamic perturbation or toxic effects occur in theorgan systems of the animals or the patients. When the level of chemicaltoxicity is minimal, it allows a large variety of usefulradiopharmaceuticals to be produced for assessing physiologic andpathophysiologic processes in tumor biology.

In vivo ^(99m)TcO₄ scintigraphy. U251MG and U87MG glioma cell lines areinjected intracranially as described above into 6-10 week old nude mice.After 5 days of tumor growth 1×10⁸ PFU of Δ24-NIS virus is injectedintracranially through the screw guide. The animals are then injectedthrough tail vein with 0.5 mCi of ^(99m)Tc-pertechnetate in 200 μl ofsterile saline. The mice are then positioned on a gamma camera with a140 keV-high resolution colimator. Images are obtained at 5, 15, 30, 60,90 and 120 min after injection with ^(99m)TcO₄ using 1 and 2 minacquisition times.

Human xenograft Models. A reproducible malignant glioma animal model isa component of the studies described herein. The U87MG model wasselected for comparing tissues and evaluating the presence of adenoviralreplication, as well as for correlating these changes with alterationsobserved using radioisotope imaging techniques. This cell line has beenwell characterized. It emulates many of the characteristics of de novohuman gliomas including response to growth factors (Pollack et al.,1990), and is readily and reproducibly used to create an intracranialmodel of human glioblastoma. The studies described herein also utilizethe U87MG model to assess if there is an improved therapeutic potentialwhen therapeutic doses of radionuclides are used in conjunction withΔ24-NIS administration. The U87MG cell line is a stable, immortalizedhuman glioblastoma cell line. U87G tumor cells readily grow in the brainof nude mice. For example, in initial studies there was a 100% tumortake in mice, which without treatment results in the need for sacrificeat 21 days (±2 days). Molecular characteristics of the U87MG modelinclude marked expression of coxsackie and adenoviral receptor (CAR), aswell as Rb, p16, and p53. The reproducibility of the intracranial animalmodel is based on the implantable screw-guide system developed byFrederick Lang, M.D., M.D. Anderson Cancer Center. In brief, a guidescrew is implanted into a small drill hole in the skull 2.5 mm laterallyand 1.0 mm anteriorly to the bregma. Tumor cells (5×10⁵) are then slowlyinjected using an automated injection system, the depth of injectioncontrolled by a collar on the syringe. Early studies showed a 97%success rate with successful delivery of agents into an establishedtumor.

Harvesting Technique. To permit optimal comparison between tissue-basedimmunohistochemistry (IHC) measures and in vivo imaging measures, theorientation of the animal's head and tumor in the axial and saggitalplanes are registered at the time of tissue harvest. At the time of IHCanalysis sections will be created from tumor blocks cut out of theseplanes so that the spatial relation to the aquired images can bemaintained. A group of animals will also be harvested for whole-animalcryo-sectioning to produce correlative autoradiography registration withthe images obtained. In the animals undergoing MRI examinations (withsubsequent sacrifice for tissue samples), the extent and orientation ofthe tumor will be carefully marked with permanent dye ink; the center ofeach scan plane will also be marked. Each animal will be sacrificed byCO₂ inhalation after adequate sedation is confirmed by the toe pinchtechnique. The tumor will be resected en bloc while maintaining carefulattention to preserving the orientation of the tumor with respect to theimaging scan plane and range. The MRI scan plane that traverses themaximum diameter of the tumor will be marked, and the tumor will besectioned into two pieces along this plane. One section of the tumorwill be embedded in OCT (Miles Inc., Elkart, Ind.), frozen in liquidnitrogen, and stored at −70° C. The other sections will be fixed informalin and embedded in paraffin. The tumor specimens resected from theanimals injected with Evans Blue will be similarly embedded in paraffin.Cell implantation and adenoviral treatment are performed as describedpreviously. Animal studies are conducted in the veterinary facilities ofM.D. Anderson Cancer Center in accordance with institutional guidelines.

Immunohistochemical Pathologic Analysis of Xenograft Tumor Sections.Animal brains are harvested, fixed in formalin, embedded in paraffin,and sections prepared after initial baking at 60° C. for 30 min. Thesections will be blocked with 0.3% H₂O₂ and 100% methanol for 30 min andrinsed in 10 mM PBS with 0.2% Triton X-100. The rinsed sections aretreated for 20 min in 1:50 Triton:PBS. The following antibodies areused: anti-hexon antibody (diluted 1:150; Chemicon, Temecula, Calif.),anti-human NIS antibody (Brahms Institute, Germany) and anti-E1A(diluted 1:200; Santa Cruz Biotech, Santa Cruz, Calif.). Sections willbe incubated with secondary antibodies at appropriate and standardconditions.

Non-viral Delivery of NIS. Non-viral delivery of NIS is accomplished byliposomal delivery by synthesis of liposome:adenovirus complexes (LAdC).Liposome (20 mM DOTAP:Chol) are synthesized and extruded through Whatmanfilters (Kent, UK) of decreasing sizes (1.0, 0.45, 0.2, and 0.1 nm). Forpreparation of LAdC, liposomes will be mixed with varying concentrationsof adenoviurs particles (10², 10³, and 10⁴) in 5% dextrose to yield afinal concentration of 4 mM liposome containing the appropriate viralparticles. Freshly prepared LAdC will be analyzed for mean particle sizeusing a N4 particle size analyzer (Coulter, Miami, Fla.).

In vitro Transduction Efficiency. Initial studies are carried out usingan adenovirus carrying a polynucleotide encoding a marker polypeptide(β-gal) to determine the transduction efficiency. Human glioma celllines U251 MG, U87MG, and human lung tumor cell lines A549, and H1299m,as well as normal fibroblasts, are seeded in 6-well plates at 5×10⁵cells/well. The following day, cells are transfected with LAdC, andAd-β-gal in serum-free medium for 3 h. Following transfection, cells arereplenished with appropriate medium and incubation continued. Cells willbe harvested at 24 and 48 h after transfection and stained forβ-galactosidase expression using the β-galactosidase staining kit(Promega, Madison, Wis.). Untransfected cells will serve as controls inthese experiments. Based on the transduction efficiency, an appropriateviral titer is determined for encapsulation in the liposome.

In Vivo Studies. In vivo studies involves the systemic delivery of LAdCin an experimental lung metastasis model. Human lung cancer cell linesH1299m and A549 are used to establish an experimental lung metastasismodel in nude mice or in SCID/Beige mice. These tumor models are wellestablished in the inventor's laboratory and routinely used. A549 andH1299m tumor cells (1×10⁶) are inoculated intravenously via the tailvein of nude mice and SCID/Beige mice, respectively, to establishexperimental lung metastasis. Treatment will be initiated at 6 to 10days after tumor cell injection at which time microscopic tumors in thelungs are established (data not shown). Treatment for macroscopic tumorswill be initiated on day 21 to 25, at which time large macroscopictumors in the lungs (A549) and other organs (H1299m) are established.Animals are divided into groups and injected intravenously with thevirus alone, liposome alone, and LAdC via tail vein (100 μl/animal). Theamount of virus to be complexed with the liposome and injected isdetermined as above. Animals are subsequently monitored for transgeneexpression in the lung tumors by imaging. This study will allow theassessment of (a) the possible use of Δ-NIS complexed to liposomes forsystemic delivery to treat disseminated metastases and (b) possibletreatment-related toxicity.

Statistical analysis. The main analytical goal of these studies is todetermine at which timepoint the signal-to-noise ratio (M_(obs)SNR) ismaximized for the two radioisotopes being examined. The signal isdefined as the number of gamma counts in the tumor/labeled tumor cellsand the noise is defined as a ratio between the number of gamma countsin the liver/the number of liver cells. Graphic displays of the mean SNRby time are to be provided for each isotope. Futhermore,likelihood-based methods are used to model the relationship between theSNR and time, and to determine the timepoint at which the SNR is thegreatest. Radioisotope uptake in tumors infected with Δ24 versus Δ24-NISare quantified through gamma camera imaging and autoradiography. Inaddition, the infected tumors are harvested at multiple time points andthe amount of radiation is normalized for the amount of radiationexpressed as counts per minute (CPM) against CPM of spleen. Theinventors propose to use 30 mice per group and provided power estimatesfor various effect sizes so that i is mean maximum SNR for iodine-133,μ₂ is the mean maximum SNR for ^(99m)TcO₄ and s is the common standarddeviation for both groups. The power estimates for the proposed samplesize use a Wilcoxon rank-sum test for various effect sizes (where effectsize=μ₁−μ₂/σ) and assume an alpha error rate of 0.05.

B. Results

The imaging component of these studies relies on the ability of NIS toconcentrate radioisotopes at a level sufficient for them to bevisualized above background. Current hNlS clones are able to concentrate^(99m)TcO₄ pertechnetate approximately 7-fold with only transienttransfection experiments in U251 MG cells. This cell line usually doesnot support very high levels of transfection (somewhere in the range of15% to 20%), and it is anticipated that the increased transfectionefficiency that is afforded by adenoviral constructs will significantlyimprove this SNR. Cell-mixing experiments with Δ24-NIS demonstrate amuch higher level of radionuclide accumulation at 1000% (10-fold) overcontrol cells at only a 10% infection rate. One potential issue ismaintainance of isotopes within the cell, typically organification ofthe anion is required. Such organification improves the accumulation ofthese isotopes. Cho et al., (2000) have shown that the U1240 glioma cellline was able to organify iodide to approximately 5%-6% within the cellscompared with approximately 10%-12% radioiodide organification incultured thyroid cells (a 50% retention would still allow 500%accumulation at a 10% rate of infection). If signal is not adequate inthe cell lines, similar washout experiments will be performed to gaugethe retention characteristics of U87MG and U251 MG cells.Retention/efflux of isotopes may also be addressed by treating animalswith sodium perchlorate 15-30 min after the isotope is given to theanimals.

Example 4 Characterize Δ24-NIS as a Therapeutic

Characterization of Δ24-NIS as a therapeutic will include assessing theeffectiveness of Δ24-NIS plus therapeutic radionuclide delivery inimproving animal survival, and assessing the dosimetry models ofradionuclide therapy of a P-emitter through imaging usingNIS-accumulated gamma emitter radionuclides.

Typically, current gene therapy methods are sub-optimal for thetreatment of cancer. In one aspect of the invention, is the improvementof oncolytic adenovirus treatment strategies, particularly forrefractory tumors. The Δ24-NIS system may be used for concentratinguseful isotopes such as ¹³¹I as well as ¹⁸⁸ReO₄. Use of the Δ24 systemwill be studied in an animal model system. Specifically, improvement inanimal survival will be assessed after infection with either Δ24 orΔ24-NIS with subsequent administration of therapeutic concentrations ofthese radioisotopes. Any potential improvement in the “bystander effect”needs to be assessed in animal models prior to future contemplation ofusing these strategies in human clinical trials. Additionally, thesestudies will also help to apply and modify current dosimetry models byusing gamma emitter isotopes for predicting accumulated dosing withintumors by NIS for eventual use of beta-emitter radioisotope therapy.

Any additive effects of oncolysis and radionuclide accumulation viaconcentration by the NIS transgene translates into improved therapy willbe assessed, particularly for the intracranial glioma mouse xenograftmodel. The endpoint will be survival and since the glioma model used hassuch a short and steep death curve, assessing modest increments inanimal survival is feasible. However, due to the uncertainty of type,dose, and timing of the radionuclides being used, the number ofcombinations, and therefore animals, could be quite large. To adequatelydeal with this large number of potential combinations and to havestatistical confidence in finding the best therapeutic combination, anadoptive randomization design will be employed. This method will limitthe number of animals needed and increase the chances of successfullyidentifying therapeutically active combinations. Additionally, matchedsets of animals will receive pure gamma-emitters at doses appropriatefor imaging but not therapy. These will simultaneously act as controlsfor tumor kill associated with radioactivity and also allow fordosimetry model confirmation.

The ability of Δ24-NIS transfected tumor cells to concentrate ¹³¹Ianalogs, ^(99m)TcO₄, Na¹²³I and ¹⁸⁸ReO₄. ^(99m)TcO₄ is a routinely usedradiopharmaceutical. Its low radiotoxicity makes it suitable for imagingand pharmacokinetic measurements. The radionuclide Na¹³¹I is usedroutinely by the inventors and is preferred for many of the studiesbecause of its short half-life and ease of use. The radionuclide ¹⁸⁸ReO₄has about 4 times the radiotoxicity of ¹³¹I and is also suitable forexternal imaging. After initial experiments to establish the efficiencyof the transfected tumor cells in animal models, treatment with^(99m)TcO₄ and Na¹²³I will be performed. To assess therapeutic dosing ofradioisotope administration to animal xenografts, studies will becarried out with Na¹³¹I and ¹⁸⁸ReO₄. Treatment efficacy will becorrelated with the theoretical dosimetry models and dosimetry measuredfrom imaging. The purpose is to determine preclinical survival advantageand pathologic changes seen in tumor samples harvested after injectionof Δ24-NIS with co-administration of ¹⁸⁸ReO₄ or Na¹³¹I.

The current tumor model of oncolytic virus transgene expression of NISin tumor cells does not provide a mechanism for organification and theproducer cells are lysed by the virus after a few days post-infection.Thus the retention time of exposure to radionuclides is transient.Because of the relatively short exposure times (about 0.5 h) comparedwith the long physical half-lives (6 to 1440 h), the relative potency ofradionuclides for therapy depends predominantly on the absorbed doserates of radiation. The dose rates, or S-values for a 1- or 2-gram tumorare provided by MIRDose in Table 1. TABLE 1 CGy/mCi-Hr (S- value) 1 gmMIRDose 2 gm MIRDose 2 gm + depth dose Tc^(99mm) O₄ 37 18 19 ¹²⁵I 46 2425 ¹²³I 66 33 — ¹²⁴I 331 179 173 ¹³¹I 393 200 195 ¹⁸⁸ReO₄ 1270 673 633

A comparison will be made with 2-gram spherical models to assess thelevel of dosage delivered to surrounding tissues. Because of this,radionuclides with long half-lives (such as I) are not as convenient forthis system compared to radionuclides such as ¹³¹I. Additionally, ¹³¹Iis a convenient agent of choice for treatment because of its readyavailability whereas ¹⁸⁸Re-perrhenate is the radionuclide with thehighest dose rate for treatment. ^(99m)TcO₄ pertechnetate is theradionuclide of choice for detection purpose because of its low doserate. As for potential toxicity from the radionuclides, the thyroid isthe critical organ for assessing the effect of radio-iodide usage.Because of the transient nature of the uptake of pertechnetate andperrhenate, the GI tract, especially the stomach will be the criticalorgan (0.2 cGy/mCi for ^(99m)TcO₄ or 6 cGy/mCi for ¹⁸⁸ReO₄).Additionally, the safety of thyroid ablation with radio-iodide treatmentis well established for patients with Grave's disease as well as forpatients with thyroid carcinoma. Doses between 100-200 mCi are routinelyused for this purpose with limited to no systemic toxicity. In factdoses as high as 400 mCi have been administired to patients with onlyminimal grade I-II hematologic toxicity being seen as the major problem.

These studies are designed to assess therapeutic advantage and survivalof tumor-implanted animals. Additionally, pathologic examination oftumors from treated animals will be analyzed at various time points. Anytreatment-induced anti-tumor effect and treatment-induced pathologicchanges to normal and surrounding brain tissue will be noted.lmmunohistochemical staining of pathologic material will use antibodiesto NIS and to late viral proteins such as hexon protein to helpdetermine viral spread and the extent of expression of NIS protein. Thisinformation will also be of value in assessing dosimetry, since depthdosimetry spheres can be calculated based on this information. A typicalcentripetal pattern of oncolytic viral spread thoughout the tumor isexpected. Centripetal spread has already been documented in tumorstreated with Δ24 adenovirus.

Dosimetry and Diagnostic Imaging with NIS. NuNu mice with transfectedtumors at different stages (different size tumors) will be injected with0.1-0.3 mCi of ^(99m)TcO₄, Na¹³¹I, or ¹⁸⁸ReO₄ and whole body images willbe obtained at 0.2, 0.5, 1, 2, 4, 6, 16, 24 and 48 h to determine thedistribution and quantity of tracer in tumors and organs. No imagingwill be performed at 24 and 48 h for ^(99m)TcO₄ due to its shorthalf-life. The effectiveness of the treatment (tumor growth curve) willbe correlated with the dose injected (higher doses for Na¹³¹I or¹⁸⁸ReO₄) and size of tumors. These results will be compared with theinventors' dosimetry models.

Animal Design and Statistical Analysis. In this randomized preclinicalstudy, mice will receive 1 of 5 doses of virus (3×10⁸, 10⁹, 10¹⁰,3×10¹⁰, 10¹¹) combined with 1 of 2 therapeutic radionuclides. Three doselevels of the radionuclides will be used, Na¹³¹I (1.0 mCi, 1.5 mCi and2.0 mCi) and ¹⁸⁸ReO₄ (1.0 mCi, 2.0 mCi and 3.0 mCi). Based on thisdesign 30 different dose/virus/radionuclide combinations and a controlarm will be used (a total of 31 groups). This high number ofcombinations will require between 240 and 300 mice to be sacrificed(assuming there are 8 to 10 mice per treatment). An alternative strategyis to use Bayesian outcome-adaptive randomization.

For purposes of this adaptive outcome design the assumption is that timeto death follows an exponential distribution (with the mean time todeath for the jth combination being λj). A vague gamma (2.70, 0.30)prior value is assumed for each λj. A maximum of 200 mice will be usedwith a maximum of 12 mice/combination. Mice will be randomized to agiven combination using the Bayesian adaptive randomization scheme. Atthe start of the trial, 4 mice will be assigned to each treatmentcombination. After the dose/radionuclide-survival data are accumulated(after 35 days) for the first 4 mice per group, the posteriorprobability pj=prob(λ, >λ₀| data) will be calculated, where λ₀ is themean time to death for the control arm. The 9 treatment combinationswith the greatest probability of being better than the control will beused. This will be followed by randomizing another 8 mice to the sametreatment combinations while randomizing 8 other mice to the controlarm. After the second stage the posterior probability for the dataaccumulated thus far in the experiment will be calculated. Once datafrom the second stage are accumulated the best twotreatment/combinations will be selected based on the value of thecalculated posterior probability. A confirmatory experiment comparingthe best two combinations with the control will be conducted.

Operating Characteristics. Based on data from studies with these mice,the survival times of control mice follow an approximate exponentialdistribution, whereas efficacious treatments follow a location-shifteddistribution. Thus to evaluate the effectiveness of the strategy whilecollecting data from the study, the inventors assumed that treatmentarms will be catetorized into three groups. The first group is the besttreatment and has only one arm. The second group is moderately effectivetreatments after 0, 3, or 6 treatments, and the last and largest groupuse placebo like substances with 23, 27, or 30 treatments for the group.Under the alternative hypothesis, the best treatment follows a shiftedexponential with distribution with expected survival time of 60 days.The moderately effective treaments follow a shifted exponentialdistribution with an expected survival of 45 days; and with control liketreatments follow an exponential distribution with an expected survivalof 30 days. These are consevative estimates as the mean survivalduration in our tumor model is 21 days with a small variance of 2.3days. The following results are obtained from 1000 simulations from eachof the scenarios mentioned above plus a null scenario where no arms aremore effective than the control. In each of the simulations although thedata were generated from distributions having mean survival timesgreater than 35 days the simulations censored obsevations to ensure thatthe simulation and the actual studies are in accord.

Following these adaptive randomization experiments the inventors willvalidate the favored combination with an additional test sets ofanimals. The best combination will be compared with the “next best” and“least best” conditions. Expectation based on projected models is thatthe inventors would have selected the best combination with aprobability of 94%.

Since efflux of the radionuclide may present the problem of sustainedexposure to therapeutic doses of a particular isotope, using areplication-competent adenovirus permits multiple treatments as thevirus propagates through the tumor. Like a “ripple” through a pond, thenext wave of viral spread provides an opportunity to re-dose withradionuclides. The inventors have extensive experience usingintracranial glioma xenograft and systemic lung cancer modes.Additionally, since the intracranial glioma xenograft model has deathcurves in a narrow range, any improvements in oncolytic effect fromtherapeutic radioisotopes will be easily identifiable. Also by using acontinuous adoptative allocation statistical model (Bayesian) a morerapid throughput and reduced number of experimental animal models willbe needed to assess multiple dosing schemes of the Δ24-NIS as well asdosing schemes for the therapeutic radioisotopes. An alternative methodwill be to assess if NIS under the control of a different promoters willincrease the therapeutic advantage. Additionally, consideration of doseadequacy will be determined assessing systemic toxicity inhistopatholgic specimens. If no toxicity is observed, additional groupsof animals will be enrolled, using an adoptive randomization scheme toassess effects from higher doses of radionuclides.

Example 5 Assessing the Imaging Capabilities of Δ24-NIS

A. Methods

These studies will track Active oncolytic spread of Δ24-NIS in a cohortof patients in a phase I clinical trial, confirm dosimetry models fortherapeutic beta-emitter administration in the phase I clinical trial byimaging NIS-accumulated gamma emitter radionuclides, and correlateimaging data in patients receiving Δ24-NIS with pathologic materialharvested after viral administration.

In an attempt to translate the imaging component of this project to ahuman clinical trial, a third cohort of patients to receive Δ24-NIS willbe added to those patients in an already approved phase I clinical trialusing Δ24-RGD. This will be coupled with administration of ^(99m)TcO₄pertechnetate for gamma camera assessment, a necessary step in theimplementation of a phase I toxicity trial to assess the propagationpotential of oncolytic viral therapy.

Based on these in vitro and in vivo data, a phase I clinical trial isdesigned that is intended (1) to provide for evaluation of the safety,tolerability, and feasibility of administering Δ24-RGD to patients withmalignant glioma, and a wide basic information about the biologic effectof injecting Δ24-RGD into human brain tumors in situ. This study hasbeen approved by M. D. Anderson's IRB. To achieve these goals, the studywillinvolve two groups of patients. A first group (Group A) will undergoa standard dose escalation study in which Δ24-RGD is administered bydirect intratumoral injection and patients are followed for clinical andradiographic toxicity. This stage will determine the maximal tolerateddose (MTD) for intratumoral injection alone, defined as one-half of alogarithm below the toxic dose. The second group (Group B) includes onlypatients with resectable tumors. These patients will first undergostereotactic injection of Δ24-RGD via a catheter that has beenpermanently implanted into the center of the tumor. After 14 days, thetumor will be resected with the catheter in place, providing a biologicspecimen for pathologic and molecular analysis. After tumor removal,Δ24-RGD will be injected into the microscopic residual tumor surroundingthe resection cavity. This will allow patients to be followed for toxiceffects of injection into a brain infiltrated with microscopic tumorcells. These patients will undergo dose escalation similar to Group Aexcept that one less level of dosage will be used for Group B than forgroup Group A. In addition to these two groups an additional cohort(Group C) will be included to evaluate the suitability of Δ24-NIS forimaging. Although the small numbers of patients will precludestatistical analysis, results from both Group A and B will providevaluable information about response and efficacy.

Under local anesthesia, a stereotactic headframe (such as the CRW orLeksell systems) will be attached to the patient. After injection withintravenous gadolinium a stereotactic MRI will be performed to localizethe tumor mass and a stereotactic biopsy will be carried out. An initialspecimen will be sent for frozen section (OCT block) to be analyzed by acertified neuropathologist to provide histologic confirmation of thepresence of tumor. A second specimen will be sent for routine fixing andH&E staining (paraffin-embedded fixed block). The third specimen will besnap frozen in liquid nitrogen.

If biopsy confirms the presence of recurrent glioma, the patient willthen undergo stereotactic-guided placement of an injection needle.Investigators will be supplied with a vial containing Δ24-RGD orΔ24-NIS. One ml containing the appropriate dose of Δ24-RGD or Δ24-NISwill be injected over 10 min at 1 to 4 sites at the surgeon'sdiscretion. The needle will be flushed with normal saline to assuredelivery of the virus (prior to needle placement the volume of theneedle must be determined so that only the 1 ml of virus and not theflushing solution is delivered). For this injection the volume will befixed regardless of tumor size.

After stereotactic injection patients will be observed in the hospitalfor a period of observation determined by the treating investigator. Anon-contrast CT will be performed to verify the injection site and toidentify any acute asymptomatic hematomas. Patients will be evaluateddaily for adverse signs and symptoms while in the hospital. Patientswill be discharged at the physician's discretion and in accordance withbiosafety standards.

Group A: Intratumoral Maximum Tolerant Dose (MTD). Cohorts of 3 patientswill receive an intratumoral injection of Δ24-RGD to determine the MTD.Cohorts of patients will be entered at each dose level as follows: DoseLevel (Δ24-RGD in pfu) 3×10⁸, 1×10⁹, 1×1¹⁰, 3×1¹⁰, or 1×10¹¹. The totalvolume of injection will depend upon the size of the tumor, but the dosewithin the volume will be fixed. The maximal injection volume for eachinjection site will be 200 μl per site, evenly distributed. If thecalculated volume is greater than 200 μl per injection, additionalinjection sites should be established. A stereotactic biopsy will becarried out, and an initial specimen will be sent for frozen section tobe analyzed by a neuropathologist to provide histologic confirmation ofthe presence of tumor. A second specimen will be sent for a routineparaffin-embedded fixed block. The third specimen will be snap frozen inliquid nitrogen.

Group B: Biologic Effects of Δ24-RGD and Maximum Tolerant Dose (MTD)After Intramural Injection. The goal of this second group is to assessthe biologic effect of Δ24-RGD within a tumor and to determine thetoxicity resulting from administering Δ24-RGD into the post-resectioncavity containing infiltrating tumor cells. To obtain a biologicspecimen and determine toxicity, a two-stage approach will beundertaken. The first stage will be a stereotactic injection of Δ24-RGDinto the tumor via an implantable permanent catheter. The second stagewill be an open craniotomy 2 weeks later with en bloc resection of thepreviously injected tumor mass (to provide a specimen that can beevaluated for Δ24-RGD effects) followed by injection of Δ24-RGD into thewalls of the resection cavity (for assessment of toxicity).

Stage 1—Δ24-RGD will be stereotactically injected using a silasticcatheter. After injection, the catheter will be cut at the level of theskull, closed with a hemoclip and be left in place until the craniotomyis performed.

Stage 2—Fourteen days after the initial stereotactic injection, a longenough time to for adequate uptake and replication of Δ-24-RGD, patientswill undergo open craniotomy (Day 15). The tumor will be removed as asingle mass with particular effort to avoid internal debulking andsuctioning of the site of prior Δ24-RGD injection. The previously placedcatheter will be used for localization of the injection sites. Aftertumor resection, Δ24-RGD will be administered by injections into thewall of the resected tumor cavity. The goal of injection is todistribute Δ24-RGD throughout the tumor wall. A grid of approximately 1cm² squares will be established. Each 1 cm² square will be injected withΔ24-RGD. Injection will be performed using a 20 g blunt tip Dandy needleattached to a 1 cc syringe. The needle will be inserted 1-2 cm withinthe parenchyma and Δ24-RGD infused for 1 min per injection. Minimalirrigation will be used after injection. The volume to be injected ateach site will be calculated as follows: Volume per injection sites=Total volume at dose level/number of injection sites.

The total volume of injection will depend upon the size of the tumor,but the dose within the volume will be fixed. The maximal injectionvolume for each injection site will be 200 μl per site evenlydistributed. If the calculated volume is greater than 200 μl perinjection, then additional injection sites should be established byadjusting the grid. During the second stage of the study, the biologicaldata obtained from stage 2 will be reviewed, and the protocol may bemodified, including but not limited to the interval between stereotacticinjection and open craniotomy, as well as the number, volume, and depthof stereotactic and open craniotomy injections.

After the craniotomy and Δ24-RGD injection, patients will be monitoredaccording to customary standards with additional biosafety levelprecautions. Patients will be evaluated daily for adverse events whilein the hospital and will be discharged at the discretion of the treatingphysician. Patients in Group 2 will not be entered into the study untilthe toxicity profile from intratumoral injection in Group A patients isestablished. To further ensure safety, Group 2 patients will not beevaluated until the cohort of patients from Group A has been analyzed.Thus, patients in Group 2 will lag behind Group 1 by at least one level.The entry criteria will be similar to a recently completed NABTC phase Iclinical trial of adenovirus p53

Group C: Intratumoral MTD with Intratumoral Imaging. The third group(Group C) includes only patients with resectable tumor as in the Group Bpatients. These patients will first undergo a stereotactic biopsyfollowed by stereotactic injection of Δ24-NIS through a catheter thathas been permanently implanted in the center of the tumor. After 14 daysthe tumor will be resected in an en-bloc with the catheter left inplace. This will provide pathologic specimens for molecular analysis andimmunohistochemical staining for assessing viral spread. After the tumoris removed, Δ24-NIS will be injected again into the microscopic residualtumors surrounding the resection cavity. The patients will then befollowed for toxic effects from the injection site intruding into theinfiltrated surrounding brain. These patients will also be followedclinically and radiographically for signs or symptoms of toxicity.Because there is concern about inter-patient variability, the inventorswill look at only one dose level corresponding to the MTD obtained fromanalysis of Group B patients. This cohort of patients (15) will be usedto evaluate the timing of M_(obs)SNR over the observational imaging dayspost-injection. The amount of virus that will be injectable will beidentical to that used for the previously described for Group B patientsand the injection of the resected cavity wall will be identical to thatpreviously described for Group B patients.

Pathologic and Imaging Correlates. By using the established en blocresection technique previously used in a p53 adenoviral glioma trial,the invenors will be able to accurately determine the extent andgeometry of active viral spread throughout the tumor bed. Thesemeasurements will be compared to nuclear imaging of patients withisotopes injected at days 3, 7, 10 and 14 prior to the 14-day surgicalresection interval. Again, analysis of tumor volume measurements at thisdose level will be compared to the imaging results from day 14 andcharacterization of this material will be related to those patientshaving M_(obs)SNR at that time point.

Statistical Analysis. The primary statistical analysis for this cohortof patients is to determine at which time point (day) thesignal-to-noise (STN) ratio is maximized (M_(obs)STN). A M_(obs)SNR willbe obtained for each patient on each day of imaging, obtained by chosingthe time point which yields the best SNR (in minutes after giving theradionuclide tracer). This measurement will be used to calculate therelative functional uptake of the radionuclide over the observed timepoints (in intervals of days post viral infection). The variable ofinterest is M_(obs)STN. The inventors plan to report the mean andstandard deviation of maximum STN by day and dose level. The generallinear model for modeling M_(obs)STN as a quadratic function of day willbe used. Because the sample size is small, an exploratory will followthe following procedure: The inventors will generate a large number ofbootstrap samples from patients' vectors of M_(obs)STNs. The M_(obs)STNwill be calculated for each day. Lastly, the proportion of times a givenday is chosen for having the maximum STN ratio will be calculated. Thisproportion approximates the probability that a given day has the highestsignal-to-noise ratio.

Operating Characteristics. The M_(obs)STN ratios of 10 or greater areexpected on the optimum day after inoculation (which presumptively willbe on day 14 for each dose level assuming 10% of the tumor cells areinfected and the potential susceptible tumor cells ate not limiting).Concomitantly a minimal STN ratio of 1 is expected for days 1 through 3(first-pass viral incubation time). Given this input, 3 simulatedscenarios will be drawn from a multivariate normal distribution with aVariance-Covariance Matrix following an AR (1) process (correlation of0.5; variance=9). In the first scenario it is assumed that the meanM_(obs)STN ratios for days 3, 7, 10, and 14 were 1, 3, 8, and 10respectively. In the second scenario it is assumed that the meanM_(obs)STN ratios were 1, 3, 10, and 8 and for the third scenario it isassumed that the mean M_(obs)STN ratios were 1, 10, 8, and 3.

B. Results

The inventors have experience in using Adp53 in a phase I clinical trialand do not anticipate problems in the proposed phase I clinical trial.M.D. Anderson Cancer Center has experience harvesting en bloc resectedtumors specimens with anatomical markers maintained along with an intactinjection catheter in place. Patients who are selected for this studywith recurrent GBM that requires surgical debulking have tumors that aresizable enough to be adequate for imaging, but it is recognize that thisgroup of patients may not be representative of all patients with tumorrecurrence who are not surgical candidates. However, given theobjectives of trying to initially visualize the tumor through the use ofΔ24-NIS, it is believe that the hypothesis can be adequately tested.

Example 6 Delta 24-Hycytosine Deaminase (A24-HYCD)

A. Materials and Methods

Cell lines and culture conditions. U87MG cells (obtained from theAmerican Type Culture Collection, Manassas, Va., cat. # HTB-14) andU251MG human glioma cell lines (kindly provided by Dr. Yung'slaboratory) were cultured in Dulbecco's modified Eagle/F12 medium (1:1,vol:vol) (Media Tech, Herndon, Va.) containing 5% fetal bovine serum(DIFCO) and 2 nM glutamine. Cells were grown in culture at 37° C. and at5% CO₂ without antibiotics and were passaged fewer than 12 times duringthe studies.

Adenoviruses. Construction of Δ24 has been described elsewhere (Fueyo,2000). This construct has a 24-bp deletion in the E1A gene (nt 923 to946, both included), a region known to be necessary for Rb proteinbinding (Whyte, 1989), corresponding to the amino acids L₁₂₂TCHEAGF₁₂₉.

To construct Δ24-hyCD, a humanized form of the nucleic acid encodingyeast cytosine deaminase (hyCD) was inserted into the E3 region of theΔ24 adenovirus. Yeast CD was choosen because of its superior enzymatickinetics over the traditional bacterial form (Hamstra, 1999). The yeastCD gene (fcy1) is derived from Saccharomyces cerevisiae and its producthas an approximate catalytic efficiency that is 280 times higher thanfrom the bacterial form of the enzyme. A series of 24 synthesized,overlapping oligonucleotide primers (Midland Certified Reagent Co.,Midland, Tex.) with pairs that were sequentially elongated by PCR wasused to construct Δ24-hyCD. The process was repeated with progressivelylonger pieces until the full-length gene was obtained. In addition, the5′ most distal oligonucleotide contained an idealized Kozac consensussequence, a proximal HindIII, and a distal XbaI restriction site forcloning. The nucleotide sequence of the synthesized polynucleotide wasalso changed significantly (102 of 460 coding base pairs) to optimize ahuman codon rather than a yeast codon preference. The full-lengthsynthesized polynucleotide was cloned into pcDNA3.1 (Invitrogen) andclones were isolated and subsequently sequenced. Several clones with theDNA sequence of interest were then transiently and stably transfectedinto U87MG and U251 MG glioma cell lines and assayed for enzymeactivity, as described below. Suitable clones expressing enzyme activitywere then cloned into the E3 region of pBHG10 (Microbix). pBHG10-hyCDand pXC1-Δ24 were cotransfected into 293 cells to allow homologousrecombination, as previously described (Fueyo, 2000).

The viruses were propagated in 293 cells and purified byultracentrifugation in a cesium chloride gradient. All viruses weretitered using a plaque method as well as optical density measurementsand were maintained at −80° C. until use. Single lots of adenovirus Δ24and adenovirus Δ24-hyCD were used in the experiments. As controls,Δ24-hyCD that had been inactivated by UV light and cells that had beenmock-infected with culture medium were used.

Chemicals. 5-FU was purchased from Sigma Chemical Company (St. Louis,Mo.) and 5-FC from SP Pharmaceuticals (Albuquerque, N. Mex.).

Real-time quantitative. PCR U251MG and U87MG cell lines were grown to95% confluence, harvested with 0.25% trypsin/EDTA, replanted into T25flasks to a total of 2×10⁶ cells, and then incubated overnight. Mediawere aspirated and 2 ml of adenovirus Δ24-hyCD was added at 0.1, 1, 5,10, or 100 pfu/cell to duplicate samples from a viral stock of 1×10¹¹pfu/ml, and the flasks were incubated for 1 h with continuous shaking.The virus was aspirated and cells were washed twice with PBS. Freshcomplete medium containing 10% FBS was replaced and the cells wereincubated at 37° C. for 24, 48, 72, or 96 h. At that time, the harvestedtissue cultures were washed twice with PBS. Floating cells were saved bycentrifugation, immediately frozen, and stored at −80° C. before themRNA was harvested. Then, cell pellets were lysed with Trizol reagent(Life Technologies) and the RNA was purified according to themanufacturer's recommendations for subsequent amplification by TaqManquantitative RT-PCR as previously described (Miller, 2002). Thefollowing primers and probe were used for the amplification anddetection of the hyCD transgene: Forward Sequence:5′-CAACATGAGGTTCCAGAAGGG-3′ (SEQ ID NO:12); Reverse Sequence:5′-CAGTTCTCCAGGGTGGAGATCT-3′ (SEQ ID NO:13); TaqMan probe:5′-TCCGCCACCCTG CACGGC-3′ (SEQ ID NO:14).

The primers were labeled with FAM label at the 5′ end and TAMRA label atthe 3′ end for yeast CD mRNA. Control primers and probes were used forthe ribosomal RNA housekeeping gene S9, a gene with little expressionvariability in human gliomas (Blanquicett, 2002). The expression of mRNAfor hyCD was quantified and reported relative to a stably expressinghyCD clone of the glioma cell line U251MG. Expression levels weredetermined with the ABI 7000 sequence detection system (AppliedBiosystems, Foster City, Calif.).

Cytosine deaminase enzymatic assays. Separation of uracil from cytosineor 5-FU from 5-FC was achieved by thin layer chromatography as modifiedfrom Rubery and Newton (1971). Briefly, aluminum-backed silica gelsheets were used (silica gel 60-F-254, EM Science, Germany). Each sheetwas spotted with a total of 5 μl of a reaction mix or standards at 1 μlsuccessive spots, with drying before additional spotting. The gel sheetswere then resolved in a chromatography tank containing a mixture of 80%chloroform and 20% methanol. The solvent front was quite rapid, with thesheets being resolved within 2-3 min. The separated cytosine and uracilor 5-FC and 5-FU were then visualized with UV excitation at 254 nm. Forquantitative enzyme assays, these resolved spots were cut out, placed inscintillation vials, and counted.

To measure enzyme activity, a procedure adapted by Hamstra et al. (1999)was used. Briefly, U251MG or U87MG glioma cells transfected with 10pfu/cell of virus were harvested after 24 h in an assay buffer (100 mMTris pH 7.8, 1 mM EDTA) and freeze-thawed 3 times. Proteinconcentrations were assessed by the Bradford method. For the conversionassay, 5-FC at 100 mM was spiked with 0.5 mM tritiated 5-FC (2 μCi/mM)and diluted at various concentrations to 30 μl reaction volumes, andeither 0.3 or 0.75 μg of protein extract was added. The reactionmixtures were allowed to incubate for 1, 5, 10, or 15 min at 37° C.,after which they were quenched by the addition of 1 M acetic acid andplaced on ice. The extent of reaction conversion was based on thefraction of produced 5-FU divided by the total counts of both the 5-FCand 5-FU bands. The percentage converted was used to calculate theproduction of 5-FU per μg of protein extract per min of reaction time.The apparent K_(m) and apparent V_(max) values were based on nonlinearregression analysis using Graph Pad's Prism program (Graph Pad Software,San Jose, Calif.). All assays were done in triplicate.

Western blot analysis. U251MG and U87MG cell lines were prepared in6-well plates and treated with Δ24, Δ24-hyCD, or PBS (mock-treatmentcondition) as described above. The cells were harvested at 24, 48, 72,or 96 h after treatment. Total cell lysates were prepared by incubatingcells with 1× SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2% w/v SDS,10% glycerol, 50 mM dithiothreitol), and protein concentration wasquantified by using the bicinchoninic acid (BCA) method (Pierce,Rockford, Ill.) and read on a Beckman spectrophotometer. Protein samples(20 μg) were boiled at 98° C. for 5 min, and lysates were separated on a15% SDS-Tris glycine polyacrylamide gel, subjected to electrophoresis at95 V for 2 h, and transferred to a nitrocellulose membrane. The membranewas blocked with 3% nonfat milk, 0.05% Tween 20, 150 mM NaCl, and 50 mMTris (pH 7.5) and incubated with primary antibody for yeast CD (1:500;Biogenesis Inc., Kingston, N.H.). The secondary antibody was horseradishperoxidase-conjugated anti-sheep IgG (Pierce, Rockford, Ill.). Themembranes were developed according to Amersham's enhancedchemiluminescence protocol (Amersham Corp., Arlington Heights, Ill.).

Cell viability assays. U251MG and U87MG cell lines were grown to 95%monolayer confluence. The cells were trypsinized and harvested with0.25% trypsin/EDTA, plated in 6-well tissue culture plates, and allowedto adhere overnight at 37° C. in 5% CO₂ humidified incubators. 5-FU or5-FC in serial 0.5-log concentrations in 100 μl aliquots was addeddirectly to the cells to achieve final concentrations, as previouslydescribed (Miller, 2002). Cells were incubated at 37° C. for 5 days andcell viability by cellular respiration was determined using3-(4,5-methylthiazole-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium)(Promega, Madison, Wis.) according to the manufacturer's protocol. Thecell survival fraction was measured at each drug concentration as theratio of absorbance at 490 nm relative to untreated cells. Thiscalculation was normalized for background absorbance of the culturemedium alone. The cell survival fraction was plotted against thelogarithm of the drug concentration, and IC₅₀ values were calculatedusing a sigmoidal dose-response curve with variable slope in GraphPadPrism 3.01.

The crystal violet assay was performed as described previously (Fueyo,2000). Briefly, cells were seeded at 10⁵ cells per well in 6-wellplates, allowed to grow for 20 h, and then infected with Δ24-hyCD, Δ24,or UV-inactivated Δ24-hyCD at 10 MOI. Either 5-FU (at 0.25 mM) or 5-FC(at 0.5 mM) was added to the cultures at different times after infection(0 to 6 days). Cell monolayers were washed with PBS and fixed andstained with 0.1% crystal violet in 20% ethanol. Excess dye was removedwith several water rinses.

In vitro cytotoxicity was quantified by using the tetrazolium salt3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)(Sigma) to measure cell viability. For this assay, 10⁴ cells were seededin 96-well microtiter plates and infected 24 h later with 0, 2, or 5pfu/cell of Δ24-hyCD or Δ24. Sixteen wells were seeded with untreatedglioma cells as a viability control, and 16 wells containing onlycomplete medium were used as a control for nonspecific dye reduction.5-FC (at 0.5 mM) or 5-FU (at 0.25 mM) was added to the cultures 1 dayafter infection. Medium was removed 7 days after Δ24-hyCD or Δ24treatment, and 100 μl/well (1 mg/ml) of MTT was added to each well. Theplates were incubated for an additional 4 h and then read on amicroplate reader at a test wavelength of 570 nm. Quadruplicate wellswere used for each condition.

Animal studies. Cell implantation and adenoviral treatment wereperformed as described previously (Lal, 2000). Briefly, implants wereplaced in 6- to 8-week old female NuNu mice by using screw-guidehardware with coordinates of 1 mm anterior and 1.2 mm lateral to thebregma. The mice were allowed to heal for 7 days, after which U87MGglioma cells were injected at a depth of 4 mm in the region of theputamen at a concentration of 5×10⁵ cells in 10 μl of PBS. Three daysafter tumor-cell implantation, a single intratumoral injection of1.5×10⁸ pfu of Δ24-hyCD, Δ24, or 10 μl of PBS was injected 3 times totalon days 3, 5, 7 for each experimental group. At 5 or 15 days after thesingle intratumoral injection, the animals were given i.p. PBS or 5-FCat 500 mg/kg once daily, Monday thru Friday, until the mice displayedsigns of neurologic dysfunction (primarily a lack of avoidance behavioror being hunched over in a posterior position) or until they werekilled. Mice were killed by CO₂ inhalation and the brains were collectedfor histopathologic examination and immunohistochemical staining. Animalstudies were conducted in the veterinary facilities of the M. D.Anderson Cancer Center in accordance with institutional guidelines.

Immunohistochemical analysis of xenograft tumor sections. Animal brainswere harvested, fixed in formalin, embedded in paraffin, and sectionswere prepared after initial baking at 60° C. for 30 min. The sectionswere blocked with 0.3% H₂O₂ and 100% methanol for 30 min and rinsed in10 mM PBS with 0.2% Triton X-100. The rinsed sections were then treatedfor 20 min in 1:50 Triton/PBS. The following antibodies were used:anti-hexon antibody (diluted 1:150; Chemicon, Temecula, Calif.),anti-yeast cytosine deaminase (diluted 1:150; Biogenesis Inc., Kingston,N.H.), and anti-E1A (diluted 1:200; Santa Cruz Biotech, Santa Cruz,Calif.). Sections were then incubated with secondary antibodies at a1:50 dilution at room temperature for 1 h. Staining was performed withacid-fast 3, 3′ amino diaminobenzidine tablets (Sigma). The sectionswere counterstained with 0.01% methanol green.

Statistical analysis. The anticancer effect in vivo was assessed byplotting survival curves according to the Kaplan-Meier method, andsurvivals among treatment groups were compared by using the logrank testin GraphPad Prism.

B. Results

The Δ24-hyCD adenovirus. To generate Δ24-hyCD the Δ24 adenovirus genome,which includes a 24-base-pair deletion in the Rb-binding region of theE1A gene (Fueyo, 2000), was modified and inserted an expressionmini-cassette in lieu of the deleted E3 region. The expression cassetteis driven by the human cytomegalovirus promoter placed immediatelyproximal to the hyCD sequence. A bovine growth hormone polyadenylationregion is immediately distal to the stop codon of the synthesized CDgene. This construct was confirmed by sequencing the mini-cassette usinga series of primers that covered the entire sequence with some overlap(data not shown). The altered nucleotide sequence that confers humancodon preference was confirmed by sequence analysis.

Production of cytosine deaminase. To determine if Δ24-hyCD expressed afunctional exogenous gene, the inventors initially assessed yeast CDmessenger (mRNA) expression by using real-time quantitative PCR. U251MGand U87MG glioma cell lines were exposed to various concentrations ofΔ24-hyCD and allowed to incubate for various periods of time Table 4.The production of mRNA was compared to the amounts of mRNA produced by aU251MG stable clone expressing hyCD (the table displays some negativevalues which reflects less mRNA production than the stable clone at theearly time points and at initial low titer experiments). A dose- andtime-dependent trend in increasing mRNA production was evident,indicating that mRNA was increasingly produced with increasing virus aswell as with longer incubation times. The production of mRNA was abatedat the longest incubation times and at the highest viral titers,probably secondary to improved viral oncolysis. In the 100 pfu/celltreatment condition, the vast majority of cells were floating at 96 h(data not shown). The U251MG cell line generated higher peakconcentrations of mRNA than U87MG cells, likely a result of their moreefficient transducibility (Miller, 2002). Production of a 2.07-logincrease in CD mRNA over that of controls was seen at only 1 pfu/celland at 72 h in the U251MG cells. This amount was approximately 10 timesthe production at 48 h (1.08-log) and is a dramatic increase over thatproduced at 24 h (0.69-log). In contrast, mRNA production in the U87MGcell line required 96 h or a titer of 100 pfu/cell to reach a 2-logincrease of mRNA production over controls. In the U251MG cell line,production of mRNA was maximal at a lower pfu/cell input dose (i.e., 1MOI) but at a long (96-h) incubation time (5.04-log) Table 4. Thisfinding further indicates that the replication competency of Δ24-hyCDdirectly influences the production of an exogenously expressed gene. Asexpected, the efficiency of hyCD mRNA production was influenced by thedifferential infectivity of cell lines and by the differential kineticsof viral production in the different cells. TABLE 2 Expression ofhumanized yeast cytosine deaminase mRNA after infection with Δ24-hyCD.Multiplicity of Infection (MOI) Cell Line Infection Times 0.1 1 5 10U251MG 24 h −0.49 0.69 1.47 1.42 48 h −2.00 1.08 1.55 1.25 96 h 2.885.04 3.96 3.93 U87MG 24 h□ −0.92 0.21 0.38 1.18 48 h −0.02 0.45 0.762.15 96 h −1.70 1.56 1.65 1.94

Consistent with the increase in the mRNA production, levels of CDprotein were also augmented in a time- and dose-related fashion. Westernblot analyses of U87MG glioma cells infected with Δ24-hyCD showed amarked increase in the amount of protein expressed relative tomock-infected U87MG controls or to U87MG cells treated with Δ24 alone(FIG. 14A). These analyses also revealed that saturation of theexpressed or translated polypeptide is limited by the lysing of cells asthe incubation times increase, in combination with large viral inputdoses. Together, these findings show that Δ24-hyCD efficientlytransduced high levels of the hyCD exogenous gene.

Next the inventors determine if exogenous hyCD protein demonstratedenzymatic activity. In initial experiments, hyCD enzyme activity wasqualitatively assessed by thin layer chromatography. No identifiableconversion of cytosine to uracil was evident in uninfected cells, afinding consistent with the lack of this pyrimidine salvage pathway inhuman cells. However, enzymatic activity in the Δ24-hyCD-treated U251MGcells rapidly converted cytosine into uracil. Enzymatic activity wasverified by using a tritiated cytosine radioisotope. Quantitativeenzymatic determination of the 5-FC-to-5-FU conversion resulted in anapparent V_(max) of 8.4 (±1.0) μM/min/mg protein and an apparent K_(m)of 0.63 (±0.04) 1/mM for hyCD-expressing U251MG cells. The enzymeactivity in crude extracts of cells infected with 10 pfu/cell Δ24-hyCDand harvested 24 h later is summarized in Table 5. The percentconversion of 5-FC to 5-FU by U87MG cells (49.7±4.8 at 5 min and90.3±6.1 at 15 min) is similar to that previously reported for thenon-humanized yCD (Kievit et al., 1999). Taken together, theseobservations indicate that Δ24-hyCD is an efficient vector that can beused to deliver an enzymatically active form of hyCD to glioma cells invitro. TABLE 3 Percentage conversion of 5-FC to 5-FU by 0.75 mg cellextract previously incubated for 24 h with 10 pfu of Δ24-hyCD per cellCell Conversion of 5-FC to 5-FU, % Type at 0 min at 5 min at 10 minK_(m) V_(max) U251MG 1.4 ± 0.9 48.6 ± 4.2 82.7 ± 5.6 0.63 ± 0.04 8.4 ±1.0 U87MG 1.3 ± 0.7 49.7 ± 4.8 90.3 ± 6.1 0.67 ± 0.05 8.3 ± 1.2V_(max) is expressed as μmol/min/mg lysate;K_(m) is expressed as the reciprocal of mM.

Increased glioma cell sensitivity to 5-FC when expressing hyCD. Todemonstrate the increased sensitivity of glioma cells expressing hyCD,U251MG cells were exposed to 5-FC at increasing concentrations andassayed for viability. Cells expressing hyCD were approximately 3 ordersof magnitude more sensitive than parental cells to 5-FC (IC₅₀, 12.4μg/ml vs. 3094 μg/ml) (FIG. 15). This increased sensitivity of gliomacells expressing hyCD suggests that Δ24-hyCD should be superior to Δ24as an antiglioma agent.

Comparison of the antiglioma effect of Δ24-hyCD and Δ24. The inventorsinfected human glioma cells with Δ24-hyCD or Δ24, with or without 5-FCor 5-FU, to analyze adenovirus- and/or drug-induced cell death. Celldeath was determined by crystal violet staining of viable cells. Theeffect of 5-FU on cell killing was no different for Δ24 versus Δ24-hyCDin U251MG (FIG. 16A) or U87MG glioma cell lines (not shown). However,the addition of 5-FC to Δ24-hyCD-transduced cells improved cell killing,presumably because of the hyCD enzymatic activity provided by Δ24-hyCD.The addition of 5-FC to Δ24-transduced cells did not affect cell killingat these incubation times and viral titers.

Next, to compare the induction of cell death after addition of 5-FC toΔ24 and Δ24-hyCD, an MTT assay was used to assess cell death and foundthat Δ24-hyCD essentially reduced the amount of virus required for cellkilling by approximately 5-fold (FIG. 16B). These findings suggest anadditive or perhaps a “bystander” effect. To accurately assess theexistence of a bystander effect, U87MG cells were infected with Δ24 orΔ24-hyCD for 1 h, the plates were washed, fresh media with 5-FC wasadded, and the conditioned medium was collected 24 h later (FIG. 16C).This medium was treated with UV to inactivate any viral activity, andthen added it to uninfected U87MG cell cultures. No effect was seen in5-FC treated cells with either conditioned medium from Δ24 or fromUV-inactivated Δ24. In contrast, cell death was evident in cells treatedwith conditioned medium from cultures treated with Δ24-hyCD+5-FC with UVinactivation. Therefore, a diffusible substance in the conditionedmedium, presumably 5-FU, was responsible for the bystander effect.

Antiglioma effect in vivo. To compare the therapeutic efficacy of Δ24and Δ24-hyCD and 5-FC in vivo, U87MG xenografts were implantedintracranially in athymic nude mice. The U87MG cell line was selectedbecause it produces glioma tumors in nude mice with highly predictablepathologic features and growth dynamics. In addition, the inventors havepreviously shown (Miller, 2002) that among three intracranial xenograftmodels of human glioma, including D54MG, U251MG, and U87MG, U87MG is themost refractory to CD/5-FC GDEPT. An implantable guide-screw systemdeveloped in the M. D. Anderson Department of Neurosurgery (Lal, 2000)was used to perform precise tumor implantation and intratumoralinjection of a therapeutic agent. A study was performed similar to thatpreviously reported (Fueyo, 2000) with 3 injections of 1×10⁸plaque-forming units (pfu). The results are shown in. Three independentstudies were performed, with 6 to 10 animals per group in eachexperiment. A significant improvement in the median survival (P<0.001)over control groups (34 and 38 days, respectively) was seen for both Δ24(55 days) and Δ24-hyCD plus 5-FC (74 days). Additionally, there was asignificant difference (P <0.002) in the median survival time betweenΔ24 and Δ24-hyCD plus 5-FC in these experimental groups.

The anticancer effect of a single injection of 1×10⁸ pfu of Δ24 wascompared with a single injection of 1×10⁸ pfu Δ24-hyCD, which isdesigned to minimize the oncolytic effect on survival by this rapidlyexpanding tumor system. Control animals were concomitantly treated withphosphate-buffered saline (PBS). Some of the treated animals were given5-FC to determine if an added benefit would result. In all experiments,the median survival time for control groups (PBS or PBS+5-FC) wasconsistently between 30 and 36 days. For animals treated with Δ24 orwith Δ24+5-FC, the median survival times were 35 and 32 days,respectively. Δ24-hyCD treatment without 5-FC led to a median survivaltime of 33 days, results which did not differ from the Δ24 or theΔ24+5-FC groups at a 0.05 level of significance. For theΔ24-hyCD-plus-5-FC condition, two 5-FC dose schedules were tested,“early” (5 days after viral injection) or “late” (15 days after viralinjection). The combined median survival time of animals treated withΔ24-hyCD+5-FC was significantly longer than the median survival ofanimals treated with Δ24 alone or with 5-FC as well as the controlanimals (P<0.0001) FIG. 17. The experiment was arbitrarily terminated atday 98 by killing the long-term survivors: 4 animals (40%) in theearly-5-FC treatment group and 1 animal (10%) from the late-5-FCtreatment group. Median survival time was no different in theearly-versus late-treated groups.

Histopathologic examination of the tumors and brains. After the micewere killed, the brains were removed, fixed in formalin, embedded inparaffin, and sectioned. Microscopic examination of control animalbrains revealed non-infiltrative tumors growing in a sphere-likepattern, with a high level of cell proliferation, hypervascularity, andabsence of necrotic areas. Lateral displacement of hemisphericstructures and collapse of the ipsilateral ventricle indicated that amass effect was responsible for the animals' deaths. The brains ofanimals that had survived for long periods, in contrast, showed completetumor regression. Tumor sequelae, including dystrophic calcification andmicrocyst formation, were identified at the tumor implantation site inthe right caudate nucleus (data not shown).

Examination of the brains of animals treated with Δ24-hyCD that diedbefore the arbitrary 98-day termination point demonstrated that deathresulted from mass effect of voluminous ellipsoid tumors. Highmagnification examination revealed a pattern of three distinct,concentric tumor zones: (1) an innermost central core consisting ofnecrosis and cellular debris (a pathologic finding that was not presentin control tumors); (2) a middle zone consisting of large numbers oftumor cells that displayed prominent viral inclusions admixed withapparently intact tumor cells; and (3) an outer zone composed of intacttumor cells with a few scattered cells showing signs of infection.Immunohistochemical analyses revealed that Δ24-hyCD adenovirus was ableto transduce late (hexon) genes, consistent with an active replicationprocess. Notably, immunohistochemical staining for hyCD showed thatΔ24-hyCD efficiently transduced exogenous hyCD protein into human gliomacells in vivo. Expression of the enzyme was detected in the cytoplasm ofthe glioma cells and correlated with cells infected with the adenoviralvector, as demonstrated by anti-yeast-CD antibody staining of gliomacells within the field of cells characterized by the presence of viralinclusion bodies. To examine the consistency of CD expression, twoΔ24-hyCD-treated animals were killed at 7 and 14 days after tumorimplantation. Immunostaining for CD was positive in the infected cellsat both time points, indicating that Δ24-hyCD was able to transduce highlevels of hyCD for at least 2 weeks after treatment in the U87MG animalmodel. Collectively, these observations demonstrate that Δ24-hyCD couldinfect and replicate in vivo and, more importantly, that Δ24-hyCD couldefficiently and consistently transduce the hyCD gene. The combination ofthese effects resulted in significantly extending survival time.

Example 7 ANG-2-Mediated Regulation of VEGF

This study investigates the mechanisms underlying the Ang-2-mediatedregulation of VEGF; to ascertain the role of Ang-2 expression in adynamic tumor model of glioma angiogenesis; and to develop an effectivetreatment based on the simultaneous targeting of Ang-2 using oncolyticadenoviruses and/or antisense-VEGF. Mechanistically, the inventors foundthat Ang-2 downmodulates HIF-1 at the posttranscriptional level andinhibits HIF-1-induced transcriptional activation of VEGF expression.Preliminary data shows Tie2 expression in glioma cells and suggests thatAng-2 function is receptor-mediated.

A. Methods

Construction and Generation of the AdAng-2 and Delta 24-Ang-2. Ang-2cDNA (1,504-bp; Gene Bank, accession # AF 004327) which are incorporatedherein by reference was amplified by RT-PCR using the following primers:5′-TACTGAAGAAAGAATGTGG-3′ (forward) (SEQ ID NO:15) and5′-TTAGAAATCTGCTGGTCGG-3′ (backward) (SEQ ID NO:16) from HUVEC cells.Subsequently, Ang-2 cDNA was cloned into an expression cassette (CMVpromoter, SV40pA) in the shuttle adenoviral vector pΔE1sp1A (MicrobixBiosystems). pΔE1sp1A-Ang-2 was cotransfected with the plasmid pJM17(Microbix Biosystems) into human embryonic kidney cell line 293 (ATCC,Rockville, Md.). After cotransfection, individual viral plaques wereisolated, and AdAng-2 identified by PCR and restriction enzymedigestion, and propagated in 293 cells. In addition, adenoviral plasmid,pBGH10-Ang-2, which has an expression cassette consisting of the CMVpromoter, Ang-2 cDNA, and the SV40-pA in the deleted-E3 region of theadenovirus, was cotransfected with pXC1-Delta-24 encompassing the 24-bpdeletion of the E1A region (923-946), corresponding to the regionrequired for Rb binding- in 293 cells (Fueyo et al., 2000). PCR andrestriction analyses confirmed the E1A deletion and the insertion of theAng-2 cDNA.

Exogenous Ang-2 expression. Western blot analysis was used to confirmthe expression of the Ang-2 protein in U-87 MG, D54 MG and U-251 MG(data not shown) glioma cell lines. Secreted Ang-2 was detected byimmunoblot analyses of conditioned media (CM) from AdAng-2 orAdCMV-treated U-87 MG cells. Exogenous Ang-2 was secreted in the media,mimicking the dynamics of the endogenous Ang-2 protein.

mRNA and protein expression. RT-PCR analysis was performed on mRNAextracted from human glioma cell lines and cultures. Primers andconditions for the PCR reaction were published previously (Poncet etal., 2003).

Cell lines: To assess the effect of overexpression of Ang-2 in vitro,the inventors selected glioma cell lines having an 80% to 100%transduction efficiency of replication-competent adenoviral vectors andexpress VEGF-A (U-87 MG, U-251 MG, LN229, SNB19, and D54 MG)(Gomez-Manzano et al., 1995). U-87 MG cells have been stably transfectedwith antisense VEGF. The D54 MG cells expressing VEGF 165 andregulatble-Ang-2 expressing cell lines were developed as described in Keet al. (2002).

Infection conditions: Infection of the cell lines will be carried out bydilution of viral stock to particular concentrations, addition of viralsolutions to cell monolayers (0.5 ml per 60 mm dish), and incubation at37° C. for 30 min with brief agitation every 5 min. This procedure willbe followed with the addition of culture medium and return the infectedcells to the 37° C. incubator.

Enzyme-linked Immunosorbent Assay (ELISA): Human VEGF ELISA analysiswill be performed to quantify secretory VEGF165 in the conditioned mediaaccording to the manufacturer's instructions (R & D Systems,Minneapolis, Minn.).

Immunoblotting, Immunoprecipitation, and Northern blot Assays: Studieswill be performed using commercial antibodies and as described inGomez-Manzano et al., 2003 and Fueyo et al., 2000.

Immunohistochemistry. VEGF-A, PCNA, αSMA will be detected byimmunostaining. Similar procedures will be used for the detection ofCD34 (Novocastra, Newcastle, UK) and Ang-2 (Santa Cruz, Calif.).Alternative in situ hybrization methods are described in Brown et al.,2000. The presence of E1A and hexon adenoviral proteins in the treatedxenografts will be assessed through immunohistochemistry.Paraffin-embedded sections from the mice tumors will be de-paraffinizedand rehydrated through xylene and ethanol into PBS. Endogenousperoxidase activity will be quenched by incubation for 30 min in 0.3%H₂O₂ in methanol. Sections will be treated with goat anti-hexon(Chemicon Inc., Temecula, Calif.) or goat anti-E1A (Santa Cruz Inc.,Santa Cruz, Calif.). Immunohistochemical staining will be performedusing diaminobenzidine according to the manufacturer's instructions withthe Vector laboratories ABC kits (Amersham).

Transcription experiments: Details on the constructs and methodology aredescribed in Gomez-Manzano et al., 2003. For mutational analyses of theHIF responsive element the methodology described in Forsythe et al.,(1996) will be followed. Briefly, the (−985) to (−939) sequencecontaining the 47-bp hypoxia response element will be amplify by PCR asa positive construct (5′-CCACAGTGCATACGTGGGCTCCAACAGGTCCTCTTCCCTCCCATGCA-3′) (SEQ ID NO:17) and using a mutated forward primer, thesame region with a 3-bp substitution (bold and underlined above will besubstituted for by AAA) will be amplified. These fragments will beinserted in the pGL2-Basic (Promega) for lucifase assays.

HIF-1 DNA-binding activity: DNA-binding activity of HIF-1α wasdetermined using an ELISA-solid phase system. Briefly, the procedure wasperformed as following: U-87 MG cultures were plated at a density of 10⁶cell/100-mm dish; 20 h later cultures were treated with AdAng-2 or theadenovirus control AdCMV-pA at a dose of 80 MOIs, or were mock-infected.Two days after treatment parallel cultures were placed in a sealedmodular incubator under hypoxic conditions (0.5% O₂) for 6 h. Nuclearextract was prepared as described in Gomez-Manzano et al. (2001). Toquantify HIF-1 activation, TransAM HIF-1 transcription factor assay kitfrom Active Motif (Carlsbard, Calif.) was used. This consists of anELISA-format assay where oligonucleotides containing a HRE motif areimmobilized in a 96-well plate. The addition of anti-HIF-1α antibody,followed by a secondary HRP-conjugated antibody was assessed byspectrophotometry.

HIF-1 protein translation assay: Cells will be plated in 6-well platesand pretreated overnight with 25 μM 2ME2 or DMSO (0.025% vol/vol). Then,medium will be changed to methionine-or cysteine-free as well as serumfree medium for 2 hrs. After this time, cells will be labeled byincubation with methionine-or cysteine-free medium containing³⁵S-methionine at a final concentration of 100 μCi/well at 37° C. forthe proposed times. Subsequently, cells will be washed twice withice-cold PBS, lysed, and subjected for immunoprecipitation usinganti-HIF antibody and protein G-agarose beads.

Endothelial cell growth assay and endothelial cell migration assay:These studies will be performed as described in Gomez-Manzano et al.,2003.

Tube Formation Assay: This assay will be performed using an in vitroangiogenesis kit (Chemicon, Temecula, Calif.) according to themanufacturer's instructions. Wells in a 96-well plate will be coatedwith ECMatrix solution, and 5×10³ cells will be plated in triplicatewells in a volume of 50 μl of EGM (Clonetics Corp.) containing 2% FBS.The cells will be incubated for 18 h at 37° C., and tube formation willbe evaluated by phase-contract microscopy. To determine the viability ofcells in these assays, cells will be stained with Hoechst 33342 (5μg/ml, Sigma) and propidium iodide (2.5 μg/ml, Sigma) for 5 min at 37°C. and analyzed by fluorescence microscopy.

Angiogenesis assay in chicken embryos. Fertilized chicken eggs (SPAFAS;Charles River Lab., Wilmington, Mass.) will be incubated at 37° C. at55% humidity for 9 days. An artificial air sac will be created over aregion containing small blood vessels in the CAM as described (Brooks etal., 1999). A small window will be cut in the shell after removing 3 mlof albumen. Filter disks (6 mm in diameter) will be coated withcortisone acetate in absolute ethanol (3 mg/mL). The CAM will be locallytreated with filter disks saturated with a solution containing bFGF (50ng/disk; R&D Systems, Minneapolis, Minn.) and VEGF121/rGel (at 1 or 10nM), rGel (at 1 or 10 nM), or buffer (PBS). The filter will be placed onthe CAM in a region with the lowest density of blood vessels, and in thevicinity, as reference, of a large vessel. Angiogenesis will bemonitored by photography 3 days after treatment. Images will be capturedusing an Olympus stereomicroscope (SZ ×12) and Spot™ Basic software(Diagnostic Instruments, Inc.). The relative vascular area will bedetermined by measuring the area taken up by blood vessels. Thisanalysis will be performed on a Macintosh computer using the publicdomain NIH Image program (available on the Internet atrsb.info.nih.gov/nih-image). The number of blood vessel branch pointswill be quantify by two researchers, and compared to the treatmentcontrols (Brooks et al., 1999).

Study of Ang-2 modification in vivo, using the chorioallantoid membrane.Data has shown that Ang-2 is responsible for disrupting the angiogenicprocess, primarily reducing branching. The inventors will study thiseffect further by stimulating angiogenesis using rhVEGfl65 and bFGF,prior to Ang-2 treatment. It will be determined if Ang-2-mediatedmodulation of the angiogenic process in the CAM model overrides the VEGFor bFGF stimulus. For branching quantification, CAMs will be picturedusing a stereomicroscope and a Sony Digital camera. Images will beanalyzed using Adobe Photoshop 6.0. and number of branching points willbe quantified. Vessel density will be quantified by using Scion Image1.63.

DNA constructs: Regulatable vector constructs are based on the BD Tet-Ongene expression system (Clontech). In certain aspects, a switchableAng-2 expression system will be developed, based on the BD Tet-OnExpression System from Clontech (Palo Alto, Calif.). A Tet-responsiveAng-2-expression cassette will be made by cloning Ang-2 cDNA into pTRE2between BamHI and EcoRV (Clontech). Cell lines will be first transfectedwith the pTet-On vector and selected clones will be then transfectedwith pTRE2-Ang-2. Clones will be selected by determining Ang-2expression under doxycycline (Dox) control, selecting optimal inductionand low background. Establishing effective concentrations of Dox will beperformed in a pilot study using 100 ng-1 μl/ml, and checking theexpression of Ang-2 after infection/drug exposure. After determining theeffective concentrations of Dox, the expression of Ang-2 will beanalyzed at different time points following drug exposure, as well as atdifferent time points following drug withdrawal.

Doxycycline administration: determination of the effective concentrationof Dox (Clontech) in isogenic U-87 MG cells will be performed in atitration experiment using different dilutions (e.g., 1, 0.1, 0.001,0.0001, and 0 μg/ml).

Engrafting Human Glioma Cells and Intratumoral Therapeutic Injection:The methodology developed by Lal et al., 2000 will be used.

Quantification of Microvessel Density (MVD). Experiments will beperformed as described in Im et al., 2001,

Quantification of PCI and MPI. At least 5 independent microscopic fieldsper tissue section will be analyzed to count PCNA-positive tumor cellsand endothelial cells. Tumor cell proliferation and endothelial cellproliferation will be quantified in vascular hot spots that areidentified by screening for areas with highest vessel density at lowmagnification. PCI will be determined by calculating the ratio betweenthe number of microvessels that colocalized endothelial cell staining(CD34) and pericyte staining (α-SMA).

TUNEL histochemistry: TUNEL histochemistry will be performed using an InSitu Cell Detection Kit, POD (Roche Diagnostics Co.).

Laser Scanning Cytometry (LSC). LSC Analysis of Tumor Microvessels isbeing described in Davis et al., 2004.

CPE assays: The MOI resulting in the destruction of 50% of the cellmonolayer (MOI50) will be determined for each virus (wild-type Ad300,UV-inactive-Ad, Delta-24, Delta-24-Ang-2, AdAng-2) in vitro. Atindicated time points post-infection, the cells are either stained withcrystal violet or photomicrographed. For staining with crystal violet,the medium is removed and cells are fixed for 3 min in 3.7% formaldehydeat room temperature. The formaldehyde is discarded, and the cells areincubated for 3 min in 1% crystal violet. After staining, the crystalviolet solution is removed, and the cells are rinsed twice in 3 ml ofwater and then air-dried. A Trypan blue exclusion test will be performedas described in Fueyo et al., 2000.

Viral Replication Experiments: Viral production will be quantified byTCID50. At the required time after infection, the cells will be scrapedinto culture medium and lysed with 3 cycles of freezing and thawing. TheTissue Culture Infection Dose50 method to determine the final viraltitration. Briefly, the cell lysates are clarified by centriftigationand the supernatants are serially diluted in medium for the infection of293 cells in 96-well plates. The cells are analyzed for CPE 10 daysafter infection. Final titers are determined as plaque-forming units,according the validation method developed by Quantum Biotechnology(Carlsbad, Calif.). Exemplary methods are described in Fueyo et al.(2003). In certain aspects, viral titers will be calculated at 48 and 96h after infection in a diverse panel of proliferating normal cells andtumor cells (Fueyo et al., 2000). These time points should test theconsistency of the ability or inability of the adenoviruses toreplicate. Viral titers will be compared between Delta-24-Ang-2,wild-type adenovirus Ad300, and Delta-24 in each cell line.

To perform U-87 MG and D54 MG intracranial implantation. The implantableguide-screw system of Lal et al. (2000), as described above, forestablishing intracranial xenografts in nude mice and for treatingengrafted tumors with intratumoral therapies (such as gene or viraltherapies) will be used.

Therapeutic index of Delta-24-Ang-2 adenovirus. The inventors willassess the effect of adding Ang-2 on the replication profile of Delta-24in gliomas in vitro. Studies will be performed in panel of glioma cells(Tie2(+): U-87 MG, D54 MG, LN229 and SNB19; Tie2(−): U251MG) and inquiescent and proliferating normal human astrocytes (NHA).

B. Results

The inventors have constructed a replication-deficient adenoviralvector, AdAng-2 that efficiently transduces Ang-2 into human gliomacells, and produced and secreted Ang-2. Expression of ectopic Ang-2results in a significant downregulation of VEGF protein and RNA levelsin U-87 MG cell line. The data derived under normoxic and hypoxicconditions indicate that this modulation occurs on the transcriptionallevel, probably by regulating HIF-Ic protein levels, and subsequentlythe DNA-binding activity of this transcription factor. Of interest isthe fact that studies revealed that not all of the glioma cell linestested were as sensitive as U-87 MG to the effect of Ang-2. For examplewhereas transfering Ang-2 to D-54 MG cells resulted in decreased VEGFand HIF-1 levels, U-251 MG cells were resistant to this effect.Demonstrating the existence of Tie2 in glioma cells, showed that thisreceptor is present in the two sensitive cell lines but was undetectablein the U-251 MG cell line. The inventors have studied the nature of thetransduction signaling that connects the Tie2 receptor to the regulationof HIF-1 activity. Data have shown that Ang-2 downmodulates p42/p44 MAPKactivity. In vitro studies showed that Ang-2 interferes with theformation of capillary-like structures by human endothelial cells.Finally, other data show that the effect of Ang-2 on VEGF expression canbe augmented by combining AdAng-2 with an adenovirus expressing anantisense VEGF cDNA.

Tie2 mRNA and protein are expressed in glioma cultures FIG. 18. A 503-bpfragment amplification was obtained from RNA extracted from HU-VEC-C,U-87 MG and D-54 MG cells; however transcript amplification was notdetected in U-251 MG cells or NIH3T3 cells (negative control).Anti-human Tie2 antibody recognized a 140-kDa band in the membraneproteins subfraction of HU-VEC-C, U-87 MG and D-54 MG, but not in U-251MG. Cytosol fraction proteins were negative for Tie2 expression.Immunoprecipitation analysis confirmed these results (data not shown).These data are consistent with the data from mRNA analyses and providecompelling evidence of the effects of Ang-2 in U-87-MG cells. Previousobservations suggest that expression of Tie2 is not exclusively inendothelial cells. Valable et al. (2003) have recently reported Tie2 RNAand protein expression in neurons, and Poncet et al. (2003) reported thereceptor expression in colon cells, colon carcinoma, and peripheralnervous system. These results are consistent with data showing thatAng-2 affects the regulation of HIF- I a activity in U-87 MG and D-54 MGcells, but not in U-251 MG.

Ang-2-mediated modulation of VEGF-A. ELISA was performed to measuresecreted VEGF protein levels in the media from Ang-2-treated U-87 MG,D54 MG and U-251 MG human glioma cell cultures. VEGF levels inAng-2-treated cells were reduced by 40% in U-87 MG and D54 MG celllines, compared with mock- and AdCMV-treated cells (P<0.001, t-test,double sided). However, no significant decrease compared tocontrol-treated cells (P>0.5, t-test, double sided) was detected inU-251 MG cells treated with Ang-2, which is correlated with the patternof Tie2 expression. In addition, transfer of Ang-2 resulted in decreasedVEGF RNA expression in U-87 MG cells compared with mock- andAdCMV-treated cells. These studies suggest that Ang-2 decreases thelevel of secreted VEGF protein at the transcriptional level and pointsto the existence of a regulatory loop between Ang-2 and VEGF.

Mechanisms of Ang-2-mediated downregulation of VEGF. Because HIF-1α isone of the most important transcriptional regulators of VEGF expression,the inventors examined its expression after transfer of Ang-2. Resultsshowed that the exogenous expression of Ang-2 in U-87 MG or D54 MGdecreased HIF-1α at the protein level. However, and consistent with nullTie2 expression, the expression of HIF-la was not significantly modifiedwhen U-251 MG were treated with AdAng-2. In addition, transcriptionexperiments using the luciferase reporter gene driven by the VEGFpromoter showed that ectopic Ang-2 decreased promoter activity when thefragment containing the HIF-1α binding site was tested. These datasuggest that Ang-2 decreases HIF-1α stimulation of VEGF expression.Northern Blot analyses showed that HIF-1α RNA levels were notsignificantly changed in AdAng-2-treated U-87 MG cells compared withmock- or AdCMV-treated cells.

Ang-2 regulation of p53 expression. Because p53 is a regulator of VEGF,the inventors determined if the transfer of Ang-2 regulates p53expression. Western blot analyses of U-87 MG human glioma cell lysates(wild-type p53) three days after treatment with AdAng-2 did not affectthe basal levels of p53 (data not shown). A second line of evidence wasprovided from Luciferase experiments, which showed that the VEGFpromoter construct containing an Sp1/p53-binding site is not sensitiveto Ang-2.

The inventors investigated the effect of Ang-2 treatment on theDNA-binding activity of HIF-1α using an ELISA-solid phase system. Underhypoxic conditions, the basal activity of HIF-1α increased (FIG. 19).HIF-DNA binding activity was attenuated in the Ang-2-treated samplescompared with mock- and AdCMV-pA-treated cultures. The effect wasobserved under normoxic and hypoxic conditions. For competitionexperiments, mutant or wild-type oligonucleotides were added to the wellprior to the addition of nuclear extract. Wild-type, but not mutant,oligonucleotide modified the activity of HIF in the samples,demonstrating the specificity of the reaction. These data are consistentwith and support our previous data, which suggest that Ang-2downmodulates VEGF levels by regulating HIF-1α.

Ang-2 inhibits Ang-1-mediated MEK/ERK phosphorylation. FIG. 21 showsAng-2 inhibits Ang-1-mediated MEK/ERK phosphorylation in gliomacultures. The Ang-2/Ang-1/Tie2 system has been studied in endothelialcells, and different pathways have been reported as involved in thecascade signaling; these include phosphotidylinositol 2-kinase (PI3-K),focal adhesion kinase, Raf/Ras/mitogen-activated protein kinase (MAPK),and Dok-R/Dok-2/Nck/Pak (Yoon et al., 2003). On the other hand,receptor-mediated HIF-1 regulation has been shown to occur viaRas/MEK/MAPK and P13K/Akt/FRAP kinase cascades (Bilton and Booker,2003). These cascades modulate protein synthesis and/or transcriptionalactivation. Initial results suggest that Ang-1 induces MEK/ERKstimulation in glioma cells. To measure MAPK activity, the inventorsmeasured the degree of phosphorylation of two MAPKs, ERK1 (p44MAPK) andERK2 (p42MAPK). Consisting with previous reports (Kim et al., 2002),rhAng-1 (100 ng/ml) increased ERK1/2 phosphorylation of HU-VEC-Cendothelial cells. Of interest, treatment of U-87 MG with rhAng-1 alsoincreased ERK1/2 phosphorylation. Co-treatment withadenoviral-transduced Ang-2 inhibited the Ang-1 induced ERK1/2phosphorylation. These data suggest that Ang-2/Ang-1 system exerts itseffect through a receptor-mediated mechanism in glioma cells.

Effect of Ang-2 overexpression on angiogenesis in vitro: These studieswere performed to elucidate the role of Ang-2 role in the process ofangiogenesis. In these experiments, HU-VEC-C treated with rhAng-2(400-800 ng/ML) for 18 h, lost their ability to form vascular-likestructures was inhibited compared with control (BSA)-treated endothelialcells. These results were not caused by decreased cell viability, asassessed by an endothelial cell growth assay performed under similarconditions. Importantly, tube formation was also compromised whenendothelial cells were incubated with conditioned media fromAdAng-2-treated U-87 MG cells, (compared to HU-VEC-C cells incubatedwith conditioned media from AdCMV-treated U-87 MG cells).

Modulation of VEGF expression by Ang-2 and antisense VEGF-A. It has beenshown that after U-87 MG malignant glioma cells were infected withAdαVEGF incorporating the cDNA of VEGF in an antisense orientation thelevel of the endogenous VEGF mRNA was reduced and the production of thetargeted secretory form of the VEGF protein was drastically decreased(Im et al., 1999). ELISA experiments were performed to analyze theeffect on secreted levels of VEGF-A from a combined treatment combiningAng-2 transfer with antisense VEGF. U-87 MG cell lines were co-infectedwith AdAng-2 (50 MOIs) and AdxVEGF (50 MOIs), and levels of secretedVEGF-A were quantified in the conditioned media. The combined treatmentof Ang-2 and αVEGF resulted in a greater decrease of secreted VEGF(33.1%), compared with the results from a single treatment (Ang-2 oraVEGF alone, 71.4% or 79%, respectively), or control (AdCMV;100 MOIs,equal to 100%).

Characterization of the modulation of VEGF by Ang-2. The VEGFs and theangiopoietins seem to play complementary and coordinated roles invascular development (Yancopoulos, 2000). Tumor cells can initially homein and grow by co-opting host vessels. However, this diversion of thehost vessels is sensed as inappropriated and the vessels regress (Holashet al., 1999a and 1999b). As vessels die, the tumor becomes secondarilyavascular and hypoxic, resulting in marked induction of tumor-derivedVEGF and robust new angiogenesis (Holash et al., 1999a and 1999b). Inthis setting, Ang-2 expression seems to correlate with vesseldestabilization, apparently leading to vessel regression in the absenceof VEGF, or robust new angiogenesis following induction of VEGF(Yancopoulos, 2000). Such a coordinated effect of VEGF and Ang-2 inangiogenesis implies certain mechanisms regulating temporally andspatially the expression of both molecules, affording their concurringor subsequential effect on tumorigenesis.

Studies conducted by the invnetors indicate that Ang-2 regulates theexpression of VEGF through transcriptional mechanisms and data supportthe fact that downregulation of HIF-1α by Ang-2 is the main factor inthe regulation of VEGF (Forsythe et al., 1996, Carmeliet and Jain,2000). The inventors have demonstrated the existence of the Tie2receptor in glioma cells, and have collected data suggesting theinvolvement of transduction signaling in the Ang-2 effect. The existenceof different levels of Tie2 in glioma cell lines offers the possibilityof 1) testing the effect of Ang-2 in two different scenarios, cellsexpressing Tie2 (Tie2+) and cells not expressing Tie2 (Tie2−), and 2)the possibility of confirming Ang-2-mediated effects in glioma celllines not only by adenoviral-transduced Ang-2, but also by treatmentsusing CM from AdAng-2-treated cells and/or direct treatment withrhAng-2.

Study of HIF-1α protein levels in glioma cells. Previous results showthat Ang-2 downregulates the protein levels of HIF-1α but not the RNAlevels in U-87 MG cells. This is consistent with the inventors currentunderstanding that the primary regulation of HIF-1α isposttranscriptional (Semenza, 2002). Preliminary data show thattreatment of Tie2 positive cells (U-87 MG and D54 MG) with AdAng-2decreased HIF-1α nuclear protein levels, but no significant decrease inHIF-1 levels was seen when U-251 MG (undetectable Tie2) was treated.These data will be expanded so that total and nuclear HIF-1α proteinlevels from several glioma cell lines will be assessed, including U-87MG, D-54 MG, LN229 and U-251 MG cells, at several time points (24 h, 48h, and 72 h) after Ang-2 transfer so that consistent results undernormoxic and hypoxic conditions (1% O₂) can be achieved. The resultswill be compared to glioma cells treated with vector control (AdCMV),vehicle control (mock), positive control (Adp53), and specificitycontrol (AdGFP); and the results will be confirmed using CM from gliomacells treated with AdAng-2 (or controls as above), or rhAng-2 (with andwithout previous stimulation of rhAng-1). For the studies using CM fromglioma cells the inventors will use transwell cluster plates to maintainco-cultures of Ad-Ang-2-treated glioma cells with untreated glioma cells(Gomez-Manzano et al., 2003). This methods allows continuous exposure ofuntreated cultures to secreted Ang-2, what is not feasible usingrhAng-2. Another variable that will be taken into consideration is theimportance of in vitro cell density, even in normoxic conditions (Shetaet al., 2001). For that reason, studies will be performed under similarcell density, and low-density cultures will be used as controls forinterpretation of the data. Also, HIF-1α RNA levels will be examined inthis panel of glioma cells to confirm previous results showing thatAng-2 dowmodulates HIF-1α at the postrancriptional level.

To obtain a better understanding of the processes involved in HIF-1αinhibition of Ang-2, the effect of Ang-2 on HIF-1α postranscritionalregulation will be studied. The Ang-2 effects on HIF-1α proteinstability by using the protein translation inhibitor cycloheximide(CHX), as describe previously (Mabjeesh et al., 2003). In the presenceof CHX, new protein synthesis is inhibited, so that HIF-1α proteinlevels predominantly reflect the degradation process of HIF-1α.AdAng-2-treated glioma cells (or treated with control adenoviruses, asabove) will be exposed to CHX for 0 to 40 min and HIF-1α protein levelswill be analyzed by Western blotting. Densitometry studies of HIF-1αprotein levels normalized with actin protein levels will allow us toobtain results on the role of Ang-2 in the stability of HIF-1α protein.

Ang-2 acceleration of HIF-1α ubiquitination and degradation.AdAng-2-treated U-87 MG cells will be studied in the presecence orabsence of the proteosome inhibitor (10 μM for 4 hours). It is expectedthat in AdCMV-treated cells, MG132 will result in enhanced HIF-1αprotein levels and multiple higher molecular weight species(poly-ubiquinated HIF-1α protein conjugates). If Ang-2 is involved inHIF-1α protein degradation, MG132 will restore the inhibitory effect ofAng-2 on HIF-1α protein levels.

Ang-2 efects on HIF-1α protein translation. U-87 MG cells will belabeled with ³⁵S methionine when treated with AdAng-2 (or controladenoviruses) for 0 to 120 min. After 15 min of labeling, HIF-1α proteinlevels will be compared by immunoprecipitation (HIF-1α antibody) andautoradiography. If Ang-2 decreases the synthesis of HIF-1α, the signalwill be higher in Ang-2-treated than in the adenovirus control-treatedcells.

Analyses of Ang-2-mediated modulation of VEGF promoter activity. Initialresults indicate that Ang-2 regulates VEGF at the transcriptional levelonly when VEGF-promoter constructs containing the HIF-1α binding siteare present, as demonstrated in U-87 MG cells. Because preliminary datasuggest that HIF-1α could be the primary mediator of Ang-2-mediatedmodulation of VEGF promoter activity, HIF-1α binding site will beinactivated in the VEGF promoter by mutation (Forsythe et al., 1996). A3-bp substitution in the HIF-1α responsive element should eliminateHIF's mediation of transcription.

Transcriptional mechanism involved in the Ang-2-mediated regulation ofHIF-1α. The Ang-2/Ang-1/Tie2 system has been studied in endothelialcells, and different pathways have been reported as being involved inthe cascade signaling; these include P13-K/Akt/mTor, FAK, Raf/Ras/MAPK,and Dok-R/Dok-2/Nck/Pak (Yoon et al., 2003). On the other hand,receptor-mediated HIF-1 regulation has been shown to occur viaRas/MEK/MAPK and PI3K/Akt/FRAP kinase cascades (Bilton and Booker,2003). These cascades modulate protein synthesis and/or transcriptionalactivation. Initial results suggest the role of Ang-2/Ang-1 to modulatethe phosphorylation status of MEK/ERK in glioma cells. To measure PI3-Kactivity, phosphorylation state of the downstream effector will beassessed, Akt, as previously described (Gomez-Manzano et al., 2003).Studies will be undertaken using adenoviral-transduced Ang-2, and alsousing rhAng-2 and CM from cells that have been treated with AdAng-2. Theinventors use established co-cultures using transwell cluster plates ofAd-Ang-2-treated glioma cells with untreated glioma cells (Gomez-Manzanoet al., 2003). This strategy will allow continuous exposure of untreatedcultures to secreted Ang-2, which is not feasible using rhAng-2.

Study of the mechanistic link between Tie2 and Ang-2 in glioma cells.Two approaches are proposed to address the importance of Tie2 in theAng-2/Ang-1 functionality. First, the phosphorylation status of Tie2receptor in glioma cells will be analyzed after rhAng-2 or CM from cellsinfected with AdAng-2, with and without previous Ang-1 stimulation. Inthe second approach, competition studies will be performed using Tie2neutralizing antibody (R&D Systems). Briefly, human glioma cells(HUV-EC-C as positive control, NIH3T3 as negative control) will betreated with Ang-1, and with Tie2 neutralizing antibody (BSA ascontrol). The phosphorylation status of MEK/ERK will be examined (aswell as the phosphorylation of Akt, it plays a role in the gliomasystem). It is comtemplated that blocking the effect of Tie2 will have asimilar result as the effect of Ang-2 on inhibiting the phosphorylationstatus of MEK/ERK. These data directly connect Ang-1 to MEK/ERK in humanglioma cells through Tie2, and indirectly will connect Ang-2 in thissystem.

Effect of the overexpression of Ang-2 on angiogenesis in vitro. Studieswill be performed on the biologic significance of Ang-2 overexpressionin human glioma cells to elucidate the role of Ang-2 in theglioma-mediated process of angiogenesis. Both human glioma and humanendothelial cells will be used. Firstly, Ang-2 will be transferred,using AdAng-2, to human glioma cells and collect the conditioned media.Then, endothelial cells will be incubated with this conditioned media,and different assays will be used to analyze their phenotypes.Conditioned media from glioma cells treated with AdCMV, AdGFP, or mocktreated, will be used as controls.

Secondly, rhAng-2 will be used to examine the effect of Ang-2 on thedynamic phenotype of endotheial cells (growth, migration, formation ofchannels) independently from other players in angiogenesis, such asVEGF. For this second approach, vehicle (BSA), rhVEGF (positivecontrol), and rhAng-1 (control for specificity) will be used ascontrols.

Endothelial growth, endothelial migration, and channel formation.Endothelial cells will be incubated with conditioned media fromAng-2-treated glioma cells to analyze endothelial growth and tubeformation assays, their migration towards conditioned media fromAng-2-treated cells will be quantified (migration assay), and themodulation of the effect of Ang-2 will be also assessed by usingstimulators of angiogenesis, such as VEGF, or by inhibiting VEGF. rhVEGFprotein and anti-human VEGF monoclonal antibody (0 to 1 mg/ml, mouseIgG2B isotype, R&D Systems) will be used as controls. In addition,studies will be performed by pretreating endothelial cells with rhAng-1,prior to the incubation of the cells with conditioned media fromglioma-treated with Ang-2. This procedure will allow one to ascertain ifAng-1 rescues the Ang-2-mediated regulation of the endothelial cellphenotype.

Ang-2-mediated effect on endothelial growth, migration, and channelformation. Because Ang-2 binds the Tie2 receptor and inhibitspro-angiogenic signals, strategies blocking Tie2 may result in an effectsimilar to that caused by the overexpression of Ang-2. The inventorswill analyze the phosphorylation status of the Tie2 receptor (comparedwith basal levels) in endothelial cells after incubating them withrhAng-2 or with conditioned media from glioma cells treated withAdAng-2. Because conditioned media from AdAng-2-treated glioma cellsshould contain low levels of VEGF protein, the phosphorylation status ofthe VEGFR-2 receptor (compared with basal levels of the protein) onendothelial cells will be assessed after being incubated withconditioned media. rhAng-1 protein will be used as positive control forTie2, and rhVEGF165 for VEGFR-2 phosphorylation analyses. Adose-dependence study will be conducted using Tie2 blocking antibody(R&D Systems) and examine Tie2's effect on the growth and migration ofendothelial cells, and on the ability of endothelial cells to formchannels in vitro. Studies will also be performed with Ang-2 and Ang-1treatment to determine if Ang-1 rescues the Ang-2-mediated regulation ofthe endothelial cell phenotype.

Construction and generation of a regulatable Ang-2 expressionadenovirus. To determine the effect of Ang-2 induction during tumorvascularization, a switchable Ang-2 expression system was developed,based on the Adeno-X Expression System from Clontech (Palo Alto,Calif.). A Tet-responsive Ang-2-expression cassette was made by cloningAng-2 cDNA into pTRE-Shuttle2 (Clontech). Recombinants were identifiedusing restriction analyses and PCR. The Ang-2 expression cassette wasexcised from pTRE-Shuttle2 and ligated to Adeno-X Viral DNA (theadenoviral genome). When the “Tet-On” expression system was employed,the expression of Ang-2 by tumor cells was induced when doxycycline isadded to the culture.

Ang-2 inhibits angiogenesis in vivo (CAM model). The inventors alsowanted to know if Ang-2 overexpression interferes with the process ofangiogenesis in vivo. For this purpose, CAMs were treated either withrhVEGF (200 ng/ml per egg) or rhAng-2 (200-800 ng/ml per egg). ControlCAMs were treated with a 0.5% BSA solution (vehicle for Ang-2). Vascularresponses were assessed 72 h later. CAMs treated with BSA displayed thetypical vascular pattern of a 12-day-old normal CAM, with thin vesselsrunning parallel to each other in a leaf-like pattern. Ang-2 treatmentdramatically decreased newly sprouting angiogenic vessels, with nohemorrhagic areas. As expected, VEGF stimulated a clearly visibleangiogenic response in CAMs (data not shown). This study suggests thatthe overexpression of Ang-2 disrupts the formation of new vessels.

Vascular development and growth kinetics in a human glioma intracranialanimal model. The inventors have performed a time point analysis of theevolution of angiogenesis in a U-87 MG animal model and establishedcorrelations between histology and growth patterns in the xenografts.The inventors have studied the Ang-2 expression pattern in thisintracranial glioma model. After intracranial injection of 5×10⁵ U-87 MGcells into the right basal ganglia of nude mice, the tumors grew from0.02 mm³ on day 4 to 100 mm³ by day 20. All animals died by day 30post-implantation. Serial temporal examination of the brains oftumor-bearing animals showed central necrosis of the xenografts within 4days of implantation. At that time, the vessels surrounding the tumordisplayed changes in morphology, including an enlarged diameter and adisorganized structure. There was also evidence of vessel interactionwith peripheral tumor cells. These vessels also showed high degree ofAng-2 expression. In addition, glioma cells were also strongly positivefor Ang-2 expression in the tumoral rim. After day 4, necrosis was notobserved. From days 15 to 20 post-implantation, the tumors were largeand hypervascularized, with large vessels. After day 20, the vesselswere numerous but thinner.

Markers for endothelial cells and peri-endothelial cells showed thatvessels were co-opted by the tumors during the early stages of theirdevelopment. Smooth muscle actin (SMA) staining was expressed inperitumoral vessels in the early stages of tumor development. However,SMA reactivity was lost in more mature tumors and in intratumoralvessels during later stages of development. Examination of the tumors atthe stage of 10-20 days revealed absence of positive vessels around thetumor for Ang-2. The intensity of Ang-2 staining in intratumoral vesselswas dramatically decreased, and Ang-2 was expressed in less than 5% ofthe tumor in glioma cells. Taken together, these data suggest that ahigh level of Ang-2 is an indicator for incipient formation of vessels,whereas low to null Ang-2 expression is required to maintain tumoralvasculature. Staining for proliferating cell nuclear antigen revealed ahigh proliferative activity ranging from a few hours after implantationto the end of the experiment. This data indicates that it is possible todelineate a well-characterized in vivo tumor model designed specificallyfor the study of antiglioma therapy.

Effect of Ang-2 expression on glioma tumorigenicity. The U-87 MG cellline was infected with AdAng-2 or an adenovirus control, AdCMV, and 3days later, cells were implanted intracranially in nude mice. Theseexperiments showed that all control-treated U-87 MG cells (n=7)developed into intracranial tumors that ultimately caused the death ofthe animals by day 35. The tumors were ellipsoid masses that compressedanatomical structures in the ipsilateral and contralateral hemispheresof the brain and were similar to those formed in other experiments usingU-87 MG cells (Fueyo et al., 2003). Conversely, all but one of theanimals bearing U-87 MG cells treated with Ang-2 (n=7) did not show anysign of general toxicity or neurological deficits by day 55 (latestpoint examined) (P=0.0001; log-rank test). In order to observe ifslow-grow tumors are arising from Ang-2-treated U-87 MG cells, theinventors have decided to carry on the study until day 100 after cellimplantation. At that moment all survivors will be euthanized and thebrains analyzed. Despite the lack of histological information on thebrain of the Ang-2 treated animals, these tumorigenicity studiesstrongly suggest that overexpression of Ang-2 resulted in inhibition oftumor production or markedly interference with the progression of tumorgrowth.

Characterization of antisense-VEGF U-87 MG cell lines. The inventorsconfirmed that the secretion of VEGF is down-modulated in these clones(anti-10: 62.8%, anti-13: 57.3%; anti-17: 71%, with respect toLacZ-transfected U87 MG cells, equal to 100%). Importantly, adenoviralinfectivity assays using AdGFP showed that U-87 antisense VEGF clonesand U-87 cells transfected with a vector control carrying LacZ,efficiently transduced GFP protein in more than 80% of the cells 3 daysafter infection with 100 MOI. This experiment showed that these cellshave the capacity to be infected by adenoviral vectors.

Effects of Ang-2 on the growth kinetics and angiogenesis in glioma. Theinventors contemplate that the VEGF blockade stage of tumor developmentis, at least in part, due to overexpression of Ang-2. The inventors willuse the U-87 MG and D54 MG models growing under different conditions ofhigh VEGF expression and low or high Ang-2, and low VEGF expression andlow or high Ang-2. Modulation of Ang-2 will be effected by use ofisogenic cell lines expressing Ang-2 under a tetracycline regulatablesystem. For modulation of VEGF levels isogenic U-87 MG cell lines willbe used: parental (high VEGF) and stably transfected with antisense VEGF(Cheng et al., 1998), as well as isogenic D-54 MG cell lines: parental(low VEGF) and stably transfected with VEGF165 cDNA. Atetracycline-regulatable system has been utilized in mammalian cells andin transgenic mice. Benjamin and Keshet (1997), among others, havetested a tetracycline-inducible system in gliomas in vivo. Using atetracycline-regulated VEGF expression system in xenografted C6 gliomacells, they were able to successfully turn on and off VEGF productionand VEGF effects. Wang et al. (2001) reported the development andtesting of a conditional expression system used to express a reportergene in human U-87 MG and SNB-19 intracranial xenografts. The xenograftsexpressed β-gal when the animals were fed drinking water containing Dox,showing that the expression of a target gene in a human intracranialxenograft can be conditionally regulated.

Conditional switching on of Ang-2 correlates with survival. A time pointanalysis of the evolution of angiogenesis in the U-87 MG animal modelhas been performed and the findings correlated with the histology andgrowth patterns of the xenografts. The data showed that it is possibleto delineate a well-characterized in vivo tumor model that is designedspecifically for the study of antiglioma therapy. Furthermore, specificexpression of different angiogenesis-related markers at different stagesof tumor growth can be used as a guide for assessing the modulation ofangiogenesis by Ang-2.

Regulatable-Ang-2 expressing U-87 MG cells will be injectedintracranially in nude mice. Three groups of animals will be establishedfor every cell line, in which Ang-2 will be turned on by administrationof Dox (antibiotic dissolved in the drinking water) through differentperiods. The particular time points selected for examining the timing ofAng-2 expression were derived from results obtained in the study of theintracranial U-87 MG xenograft. Thus, three group of animals will beanalyzed in which the expression of Ang-2 will be turn on at differenttimes: (A) the same day of cell implantation (tumorigenicity study); (B)4 days after cell implantation (angiogenesis switching; earlyvasculature); or (C) 10 days after cell implantation (establishment ofintratumoral vasculature). Animals will be sacrificed if and when theydemonstrate signs of general toxicity, or neurological signs or, in thecase of long survivors, animals will be sacrified 60 dayspost-implantation, and brains will be extracted. Similar experimentswill be performed in the D54 MG cell line.

Several reports have shown that Ang-2 promoted blood vesseldestabilization and regression in the absence of the VEGF or bFGFsurvival factors. To examine if the effect of Ang-2 in this gliomasystem is dependent on a VEGF background, in vivo studies will beperformed using regulatable-Ang-2 expressing clones from two isogenicU-87 MG cell lines that express different levels of VEGF (A) parentalU-87 MG, and U-87 MG with low VEGF expression (stably transfected withantisense VEGF), as well as with two isogenic D54 MG cell linesexpressing different levels of VEGF or (B) parental D54 MG, and D54 MGwith a high expression of VEGF165 (stably transfected with VEGF165 cDNA,(Ke et al., 2000). Statistical analyses will analyze if changes in VEGFexpression modified the effect of Ang-2 on the survival of U-87- or D54MG tumor-bearing animals. Tumors will be examined using H/E staining andimmunohistochemistry, as described herein.

Modulation of tumor angiogenesis by Ang-2. In each treatment group tumorcells will be analyzed for PCNA (proliferation marker), apoptosis(TUNEL), VEGF, and Ang-2. The expression of CD34,% TUNEL (ratioCD34/TUNEL-positive cells), SMA (pericytes) will also be determined.After exogenous Ang-2 is expressed, it is anticipated that theproduction of smaller tumors as compared to the control group. In theseAng-2-overexpressing tumors, an inverse correlation is expected betweenproliferation and apoptosis index. It is predicted that tumor cellproduction of VEGF will be significantly reduced and that microvasculardensity (MVD) (measured by staining with antibodies against CD34) willbe inversely proportional to the expression of Ang-2 and directlyproportional to the expression of VEGF. The microvessel pericytecoverage index (vessels positive for both endothelial and pericytemarkers) will be inversely related to Ang-2 expression, indicating thatoverexpression of Ang-2 destabilized vessel formation. The endpoints areto correlate overexpression of Ang-2 with (a) decreased production oftumor angiogenesis; (b) decreased tumor proliferation; (c) increasedtumor and endothelial cell apoptosis; (d) decreased formation of maturevessels; and (e) reduced tumor production of VEGF. Quantification of thedata obtained will be by three-color immunofluorescence analysis of CD31and TUNEL quantified by LSC (CompuCyte Corporation, Cambridge, Mass.)(Davis et al., 2004). The LSC is an instrument designed to enablefluorescence-based quantitative measurements on cellular preparations atthe single cell level.

The objectives include the collection of data on the timing of Ang-2overexpression and the response to that (survival) in each cell line.The studies will be performed to analyze survival in glioma-bearing nudemice with and without Dox treatment. Two isogenic U-87 MG cell lineswill be used for these studies that have been stable transfected withthe regulatable Ang-2 system. Survival curves will be estimated usingthe Kaplan-Meier method. Cox proportional hazards regression analysiswill be used to estimate the hazard ratio between groups along with a95% confidence interval for this ratio and a likelihood ratio p-valuefor testing if the ratio is different from 1 (the value of the ratio ifthe groups have the same survival distributions). The hazard ratioquantifies the relative rates of death between the groups. Based onhistoric data it is expected that the control animals will have a mediansurvival of 20 days. One aspect of these data is that there is aguaranteed time during which animals have an extremely low risk ofdeath. Based on historic data, a good estimate for this guaranteed timeis about 15 days. For a one-sided alpha of 5% and a power of 90% we need10 animals for each treatment group (10 animals×2 isogenic cell lines×2different treatments (dox)=40 animals).

Ang-2 and/or VEGF-based antiangiogenesis therapy. In addition tomechanistic obstacles, the efficiency of gene therapy in general, hasbeen halted by the inability of the replication-deficient adenovirus totransduce a sufficient number of cells to induce a significantanticancer effect in vivo. Adenovirus typically delivers a functionalp53 protein in the vicinity of the injection site, but that the majorityof the tumor remains uninfected (Lang et al., 2003). For that reason,replication-competent adenoviral system will be used as a delivery tool,preferably Delta-24 adenovirus, which replicates efficiently in humanglioma cells but does not replicate in normal cells (Fueyo et al.,2000).

Although the selectively and safety of the oncolytic system in vitro andin vivo have been demonstrated by our laboratory (Fueyo et al., 2000,Suzuki et al., 2001), the oncolytic adenovirus by itself has been unableto suppress tumor growth in most of the animals. For that reason, anoncolytic adenovirus that will deliver high levels of Ang-2 to thetumor. A pilot study showed that the new construct also replicates andexpresses Ang-2 at high levels in an in vivo intracranial glioma animalmodel. The results of that experiment also suggested that Delta-24-Ang-2has an anticancer effect. Finally, data show the effect of otherantiangiogenic agents that are in clinical trials or are being plannedfor testing in clinical trials for patients with malignant gliomas.

Transfer of antisense-VEGF inhibits tumor growth in vivo. VEGF ispotentially an optimal target for therapeutic strategies because it isessential for tumor growth and progression. The recombinant adenoviralvector Ad5CMV-αVEGF incorporates the coding sequence of wild-typeVEGF165 cDNA in an antisense orientation. Infection of U-87 MG malignantglioma cells with AdαVEGF reduced the amount of endogenous VEGF mRNA anddrastically decreased the production of the targeted secretory form ofthe VEGF protein. Subcutaneous human glioma tumors established in nudemice were treated with an intralesional injection of AdαVEGF, resultingin inhibited tumor growth (P=0.004). Taken together, these findingsindicated that the efficient downregulation of VEGF produced by tumoralcells using antisense strategies produces an antitumor effect in vivo.

Transfer of Ang-2 improves survival of intracranial U-87 MGtumor-bearing mice. Intratumoral treatment with Ang-2 was assessed formodification of the survival of mice bearing U-87 MG intracranialtumors. The inventors implanted 5×10⁵ U-87 MG human glioma cellsintracranially into nude mice. Three days later, treatment with 1.5×10⁸p.f.u. of AdAng-2 or AdCMV was injected into the tumor using aguide-screw system. Treatment was repeated twice weekly until mice,whose tumors were treated with AdCMV, died (day 32). In contrast, 50days after cell implantation, 70% of the animals whose tumors weretreated with AdAng-2 were still alive, without showing any signs ofgeneral or local toxicity. These experiments demonstrated asignificantly improved survival of animals treated with AdAng-2 comparedwith AdCMV-treated animals (P=0.0001, log-rank test). A secondexperiment performed with a decrease in the number of intratumoralinjections administered (until day 20 post-cell implantation) alsoresulted in a significant increased in survival (P=0.012; log-ranktest). However, the fact that a comparative study between bothtreatments showed a significant difference in the improvement in themedian survival vs. control for the experiment with longer period oftreatment compared with the three-weeks period treatment (P=0.026;log-logistic distribution), lead to the thought that the continuousexpression of Ang-2 is necessary to obtain an anticancer effect. Forthat reason, the combination of oncolytic adenovirus continuouslyexpressing the Ang-2 transgene is especially interesting.

Armed Delta-24-Ang-2 efficiently transduces Ang-2 in vitro. Initialresults support the use of the replication-selective oncolyticadenovirus Delta-24 as an expression vector. U-87 MG cell were infectedwith the replication-deficient AdAng-2 or with the virus controlAdCMV-pA at 5 MOIs, or the new construct Delta-24-Ang-2 at 0.1 MOIs.Cell lysates were collected 2 and 4 days after infection and equalamount of proteins were analyzed by Western Blotting for expression ofAng-2. The replication-competent adenovirus Delta-24-Ang-2 transducedhigher levels of Ang-2 than its replication deficient counterpartAdAng-2.

Anticancer effect of Delta-24-Ang-2 in vitro. In this study U-87 MGhuman glioma cells were infected with Delta-24-Ang-2, or WV-inactivatedadenovirus (at doses of 0, 0.1, 1, 5, and 10 MOIs). Crystal violetviability assays showed that Delta-24-Ang-2 exhibited an increasing CPEwith increasing MOIs. The oncolytic effect was noticeable with a 1-MOIdose and was higher than 100% with a dose of 10 MOIs on the 8th dayafter infection. To ascertain if the CPE was due to adenovirusreplication, TCID50 replication assay was performed. Three days afterthe infection of glioma cells with 1 MOI of Delta-24-Ang-2, media andcell lysates were collected and the titer of adenovirus was measured in293 cells. No new viral progeny were detected in cells infected withUV-inactivated adenovirus. Two independent experiments showed thatDelta-24-Ang-2 replicated in U-87 MG cells (3.1×10⁶ pfu/ml, and 7.9×10⁶pfu/ml). Studies are in progress to compare replication and CPE inducedby Delta-24-Ang-2 to Delta-24 and wild-type adenoviruses. Of importance,the above studies suggested that Delta-24-Ang-2 maintains itsreplication phenotype and is able to transduce Ang-2, at higher levelsthat a replication deficient vector.

Delta-24 expresses Ang-2 and replicates in vivo. 5×10⁵ U-87 MG humanglioma cells were injected intracranially into nude mice. Three dayslater 1.5×10⁸ pfu of Delta-24-Ang-2 (2 mice) or UV-inactivated-Delta-24-Ang-2 (2 mice) were injected into the tumor using aguide-screw system. Animals were sacrified 11 days after implantationand brains were collected. Fresh sections were stained with H&E, andpermanent paraffin sections were deparaffinized and stained to detectthe expression of Ang-2. Optical microscopy of the UV-inactivatedDelta-24-Ang-2 treated tumors showed a tumoral mass (>20 mm3) that washighly vascularized and that had no areas of necrosis. In contrast,Delta-24-Ang-2 tumors were significantly smaller (<0.5 mm³), and showednecrotic areas. Immunohistochemical staining of Ang-2 in U-87 MG humantumor xenografts treated with Delta-24-Ang-2, demonstrated high levelsof transduced protein compared with the control-treated xenograft. Ofimportance, staining with anti-hexon protein (structural viral protein)was positive in xenograft treated with Delta-24-Ang-2, suggestingadenoviral replication. These studied suggest that the Ang-2-armedDelta-24 adenovirus transduces high levels of Ang-2 and induces animportant suppression of glioma growth in vivo.

Co-infection with Delta-24 enhances the potency of replication deficientvectors. U-87 MG cell line was infected with Delta-24 (10 MOIs) orUV-inactivated Delta-24 (10 MOIs), and the replication-deficient AdGFP(25 MOIs) or the control AdCMV (no exogenous cDNA). Three days latercultures were imaged under a fluorescence microscope. Preliminary datashowed an increase of positive-green cells when AdGFP co-existed withDelta-24, suggesting that Delta-24 acts as a facilitator for thedelivery of replication-deficient E1-deleted adenovirus vectors(Steinwaerder et al., 2002; Bemt et al.; 2002, Carlson et al., 2002;Alemany et al., 1999).

LN229 and SNB19 human cell lines exhibit invasive phenotype in vivo. Tocomplement the U-87 MG in vivo system, several human glioma cell lineshave been tested that will have more clinical significance. In thisregard, LN229 and SNB19 human glioma cell lines are tumorogenic wheninjected intracranially and exhibit an invasive phenotype in vivo.Furthermore, both cell lines expressed Tie2, which makes them likelycandidates for an Ang-2-based strategy.

Testing the effect of antiangiogenic compounds on the survival of humanglioma xenograft mouse model. Comparative survival analysis of theanticancer effect of different antiangiogenic treatments have beenperformed using the U-87 MG xenograft mouse model). PTK787/ZK 222584 wasevaluated as a VEGFR inhibitor (Thomas et al., 2003); andimatinib/Glivec as a potent PDGFRP inhibitor (Manley et al., 2002).Briefly, human glioma cells (5×10⁵ U-87 MG) were engrafted into thecaudate nucleus of athymic mice using a guide-screw system as previouslydescribed (Fueyo et al., 2003). PTK787 or Imatinib was administered at adose of 25 mg/kg/ip/day or 20 mg/kg/ip/twice/day, respectively, untilthe animals showed signs of general or local tocixity. Treatment withthese drugs did not significantly improve the survival of these animals(P>0.5, logrank test) compared with control-treated animals (vehicle).Neither group treatment had long-term survivors.

The antiglioma effect of combined therapies based on the transfer ofAngiopoietin-2, using oncolytic adenoviruses, and antisense VEGF. Theinventors have designed an Ang-2/VEGF-based antiangiogenesis therapywith tumor selectivity and efficient delivery. In addition tomechanistic obstacles, the efficiency of gene therapy is abrogated bythe inability of replication-deficient adenoviruses to transduce anumber of cells in vivo sufficient to induce a significant anticancereffect. This limitation is underscored by results obtained in a clinicaltrial using Ad-p53 in gliomas. This trial demonstrated that theadenovirus delivered a functional p53 protein in the vicinity of theinjection site, with the majority of tumor cells remaining uninfected(Lang et al., 2003). Because of that finding, we will utilize areplication-competent adenoviral system as a delivery tool in thiscompetitive renewal. The system is based on a replication-selectiveDelta-24 (Fueyo et al., 2000).

Role of the Rb pathway in controlling the oncolytic effect ofDelta-24-Ang-2. Since Delta-24 and Delta-24-Ang-2 encompass a similarE1A deletion, it is expected that oncolysis can be controlled byrestoring the Rb pathway. To demonstrate this the ability ofDelta-24-Ang-2 to replicate in cancer cells expressing null or restoredRb function will be assessed (Fueyo et al., 2000).

The effect of Delta-24 on HIF-1α activity. Zoltan et al. (1996) reportedthat in response to hypoxia, E1A inhibits HIF-1 activity by specificallytargeting p300. That phenomenon could have synergistic/additive effectswhen Delta-24 is combined with the expression of Ang-2. From amechanistic point of view the effect of E1A could interfere withinterpreting the effect of Ang-2 on the activity of HIF-1α. Theinventors will assess the effect of oncolytic replication competentadenoviruses on two different systems. The first approach will involvethe use of normal rodent cells, NIH3T3 (ATCC), which do not support thereplication of the adenovirus but allow for the expression of E1A. Thiscellular context will allow the dissection of the effect of E1A onHIF-10 activity, independently from other adenoviral proteins and fromadenovirus replication. The second approach will involve the use ofglioma cultures (U-87 MG), which have constitutively high levels ofHIF-1. After infecting both cultures with Ang-2 under hypoxic conditions(Zoltan et al., 1996), HIF activity will be assessed by detection ofluciferase to quantify the transcriptional activity of HIF-1 and itsDNA-binding capacity. If Delta-24 downregulates HIF-1 activity,additional oncolytic adenovirus will be studied. One of these oncolyticadenovirus incorporates a deletion that encompasses the p300-bindingregion of E1A (Delta-39; aa. D₄₈LDVTAPEDPNEE₆₀) (SEQ ID NO:18). Theother adenoviral construct has a combination of the E1A deletionspresent in both Delta-24 and Delta-39 (Delta-24/39) constructs. If themutant Delta-39 does not have any effect on HIF-1α activity, anexpression cassette of Ang-2 will be inserted in the E3 region of theDelta-24/39. Thus, the tumor selectivity produced by the 24-bp deletionwill be preserved and will have impaired the ability of E1A to interactwith p300.

Delta-24-Ang-2 replication in vivo and modification of the angiogenicprocess. 5×10⁵ human glioma cells (U-87 MG and LN229) will be injectedintracranially into 5 nude mice. Three days later, 1.5×10⁸plaque-forming units of Delta-24-Ang-2 or UV-inactivated-Delta-24-Ang-2will be injected into the tumor using a guide-screw system. Animals willbe sacrificed 20 days after treatment to examine the spread of theDelta-24-Ang-2 adenovirus within the tumor. Fresh sections will bestained with H&E. Permanent paraffin sections from the brain will bedeparaffinized and stained for the detection of viral proteins such ashexon and E1A. Angiogenesis analyses will be performed. Tumor size,necrosis areas, MVD, PCI, endothelial or tumor apoptosis, and expressionand localization of Ang-2 and VEGF will be determined.

AdαVEGF replicative properties when it co-exists with Delta-24.Infection of Delta-24 with AdGFP (has a similar structure as AdαVEGF,but with GFP cDNA) (Fueyo et al., 2000) will allow us to indirectlyanalyze the replication of the adenoviral vector. U-87 MG, D54 MG, andLN229 cells will be infected with Delta-24 at an MOI of 10(UV-inactivated Delta-24 as control) and AdGFP (AdCMV as control) at anMOI of 25 or 50. Two and four days later GFP-positive cells will bescored in a total of 500 cells.

Infection of Delta-24 with AdαVEGF will allow one to directly analyzethe replication of the adenoviral vector. U-87 MG, D54 MG, and LN229cells will be infected with Delta-24 at an MOI of 10 (UV-inactivatedDelta-24 as control) and AdαVEGF (AdCMV as control) at an MOI of 25 or50. Two, four, and six days later, quantitative PCR amplification of afragment of the AdαVEGF containing the insert will be performed.

In vivo anticancer effect of combining Delta-24-Ang-2 with AdαVEGF.Studies will be performed in U-87 MG, D54 MG, and LN229-basedintracranial xenografts in nude mice. Treatments will consist ofintratumoral administration of Delta-24-Ang-2 with AdαVEGF,Delta-24-Ang-2 with AdCMV, UV-inactivated Delta-24-Ang-2 with AdαVEGF,UV-inactivated Delta-24-Ang-2 with AdCMV, wild-type Ad300 with AdαVEGF,and Ad300 with AdCMV. Results from our preliminary experiments show thatan empirically chosen dose of 1.5×10⁸ pfu (administered 3 times)significantly improves survival. For that reason, we selected this asthe initial dose to test in the animal model.

Although the primary studies will be focused on survival, at the momentof death (cancer-induced or sacrificed), all of the brain tissue will beextracted. The tumors will then be examined using H&E staining andimmunohistochemistry for viral proteins and angiogenesis. Since therewill be only one time point, the pathologic examination will be utilizedfor a descriptive analysis.

Although treating U-87 MG intracranial xenografts with Delta-24 resultsin an increased survival, 80% of the animals died from mass effect ofthe tumor growth. Since tumor growth is, at least in part, related toincreased angiogenesis, the inventors expect that after combiningDelta-24 with the delivery of Ang-2, along with a decrease in VEGFlevels, the percentage of long-term survivors will increase.Delta-24-Ang-2 may be used in combination with other antiangiogenicagents to abolished mature, preexisting vasculature in establishedtumors.

Example 8 Tropism of Delta -24 Adenovirus to Human Glioma Cells

A. Material and Methods

Delta-24-300. pXC1-D24 containing the D-24 deletion (Fueyo et al., 2000)was used as the template DNA. Two 5′ phosphorylated primers consistingof sequences of 20 nucleotides on either side of the deletion (sequenceDLDVTAPEDPNEE, 48-60 aa of E1A protein (SEQ ID NO:18)) were used forlinear mutagenesis primer-incorporating PCR. The template was linearizedand the PCR product was recircularized by ligation and transformed intoE. coli to produce the vector pXC1-D24/300 encompassing deletionsdisabling binding of E1A protein with both Rb and p300. pXC1-D24/300 andpBHG10 (Microbix Biosystem, Inc.) were co-transfected byliposome-mediated method into 293 cells for homologous recombination andindividual plaques were isolated and amplified.

Anti-glioma Effect of Delta-24-300 U-87 MG, U-251 MG and D54 MG humanglioma cells were infected with Delta-24-300, Ad300 (wild-typeadenovirus), Delta-300, Delta-24, or UV-inactivated Ad300 and assessedcell viability by crystal violet assay and then quantified with MTTtests. Both analyses showed a consistent dose-response effect ofDelta-24-300 on the three human glioma cell lines. MTT analyses furthershowed that the decreased viability observed in the crystal violetassays was highly reproducible and dose dependent in the three celllines tested. Delta-24-300 is able to induce cell death in glioma cellsin vitro at a dose of less than 10 MOI and causes a 50% decrease is cellviability.

Differential Expression of Viral Proteins in Glioma and NormalAstrocytes Expression of early (E1A) and late (hexon) genes wereevaluated in U-251 MG and normal human astrocytes prepared 16 hoursafter infection with Delta-24-300, Delta-24 or Delta-300. Expression ofE1A and fiber protein was significantly down-regulated in theDelta-24-300-treated NHA as compared to the Delta-24-300-treated U-251MG glioma cells.

Replication Profile of Delta-24-300 in NHA Cultures Delta-24 has beenshown to replicate inefficiently and wild-type adenovirus replicatesefficiently in quiescent normal cultures (Fueyo et al., 2000, 2003). AnE2F-1-promoter construct driving the reported luciferase gene (Johnsonet al., 1994) is used to determine if adenoviral-mediated S-phaseinduction would be impaired following infection of proliferating NHAwith Delta-24-300 while wild-type adenovirus would efficiently induce Sphase. E2F-1-activity in NHA cultures infected with Delta-24-300 wassimilar to the mock-treated cultures and lower than that induced byDelta-24. As expected, the highest E2F-1 promoter activity was observedin cultures infected with the wild-type adenovirus. The data suggestthat Delta-24-300 is unable to elicit an S-phase like response in normalcells and suggest that its toxicity will be lower than that of Delta-24.

To confirm the selectivity of Delta-24-300, the replication phenotypesin glioma cultures and actively-dividing NHA infected with Delta-24-300,Delta-24, Delta-300, wild-type or UV-inactivated wild-type adenoviruswere compared. Analysis of viral titers 3 days after infection revealedthat the replicative ability of Delta-24-300 is greatly attenuated inthe dividing normal cell population and that the ability of Delta-24-300to acquire a replication phenotype in normal dividing cells is impairedcompared to Delta-24 or Delta-300.

Construction of Delta-24-vIII. Delta 24-vIII contains Delta-24 deletionand the chimeric fiber targeting EGFR vIII. The chimeric fiber wasdesigned having the N-terminal of AdS fiber protein (1-83aa), T4febritin (bacteriophage T4 fibritin halican domain and fold (233-487aa), linker (e.g., G4SG4SG4S linker), and PEPHC1 ligand (HFLIIGFMRRALCGA(SEQ ID NO.:19)) (Krasnykh et al. 2001; Campa et al. 2000). The tail andT4 fibritin moieties ensure the formation of the trimeric structure ofthe fiber as well as the correct insertion of the fiber into the virionparticle through the tail. The linker joins the fiber and the anti-EGFRvIII ligand.

To construct the chimeric fiber, oligonucleotides of cDNAs for thelinker and ligand (PEPHC1) were synthesized according to their aminoacid sequences. Complementary oligonucleotides were annealed andinserted into plasmids. The pQE-trisystem (Qiagen, Valencia, Calif.) wasused to construct the chimeric fiber so that the protein can ultimatelybe expressed in E. coli or mammalian cells to verify fibertrimerization. Trimerization was assessed by expression in E. coli M15(pREP4) (Qiagen) and human cancer cells. The proteins collected from theE. coli or human cancer cells were denatured or not denatured, and thenseparated by SDS-PAGE and immunoblotted with anti-Ad fiber tail MAb 4D2(NeoMarkers, Fremont, Calif.). The protein was successfully able totrimerize, showing a shift in the non-denatured sample to approximatelythreefold the size of the denatured sample (data not shown).

Generation of D24-30bvIII. The whole fiber cDNA (1745 bp) has beendeleted from pAB26 (Microbix Biosytem, Ontario, Canada) and created aPac I site at the position where the chimeric fiber will be inserted.Three deletions (fiber, Delta-24 and p300) will be made within the BstB1/Xba I fragment of the pFG173 plasmid (Microbix Biosytem, Ontario,Canada). In particular, these deletions will be made in a pBluescriptKS+ backbone and the Bst BI/Xba I fragment containing the deletions willbe ligated back to the remaining of pFG173 to obtain pFGΔfΔ24-300. Atthe same time, an expression minicassette for Enhanced GreenFluorescence Protein (EGFP) will be inserted into the deleted E3 regionof the adenovirus and will be used as a reporter of adenoviral infectionability. The EGFP expression cassette will also be inserted intomultiple cloning sites of the plasmids pAB26 and pABΔf to yield pAB-EGFPand pABΔf-EGFP.

Construction of the shuttle plasmid containing the chimeric fiber. Thechimeric fiber FFL, which contains the peptide targeting EGFRvIII, willbe inserted into pABΔf or pABΔf-EGFP in the same direction as theoriginal fiber to obtain pAB-FFL or pAB-FFL-EGFP. Similarly, FF6H (achimeric fiber containing a peptide control) will also be cloned intopABΔf or pABΔf-EGFP to yield pAB-FF6H or pAB-FF6H-EGFP.

The shuttle plasmids and pFGΔfΔ24 will be co-transfected into 211B cellswhich constitutively express fiber protein to allow homologousrecombination. The media from the co-transfected 211B cells suspected ofdemonstrating cytopathic effect will be collected and tested via a PCRassay to verify the recombinant viral genomic region for the specificvirus. The confirmed recombinant virus from the cell lysates will beplaqued and each individual plaque will be amplified and verified in211B cells. At this stage, the virions will have both the Ad5 fiber andchimeric fiber. The modified adenoviruses will be propagated in A549cells (ATCC) where the virions should have only chimeric fibers. SinceA549 cells do not express adenoviral genes, the production of wild-typeadenovirus is highly unlikely. PCR amplification of E1A followed byenzyme digestion (Fueyo et al., 2000) will be performed to detect theE1A mutation for each batch of the virus.

EGFR and EGFRvIII Expression in U87MG.wtEGFR and U87 MG.ΔEGFR Cells Totest the ability of the new virus to target EGFRvIII, U87 MG.wtEGFR andU87MG.ΔEGFR cell lines were obatined (Nishikawa et al. 1994; Mishima etal. 2001). These cell lines stably over-express either wild-type EGFR(U87MG.wtEGFR) or EGFR vIII (U87MG.ΔEGFR). EGFRs were detected byimmunoblotting and immunohistochemistry. The anti-EGFR antibody (CellSignaling, Beverly, Mass.) recognizes both EGFR and EGFRvIII and theanti-EGFRvIII antibody (Zymed Laboratories Inc., San Francisco, Calif.)specifically recognizes EGFRvIII. These cell lines represent a bona fidesystem to test selectivity of the targeted adenovirus infectivity andwill be used for in vitro and in vivo studies.

Expression of EGFR and EGFRvIII in Heterotransplants from tumors.Heterotransplants of human gliomas expressing EGFR or EGFRvIII will beused to study the targeting of the adenoviral constructs.irrunohistochemical analyses detected expression of EGFR and EGFRvIII.Interestingly, the heterotransplants exhibited an invasive pattern wheninjected intracranially in nude mice.

Infectivity of Mesenchymal Stem Cells. The ability of adenovirus toinfect human stem cells, will be assessed using human mesenchymal stemcells and two GFP-adenoviral vectors. One of the vectors was redirectedto infect through a CAR-independent pathway. Studies showed thatadenovirus poorly infects mesenchymal stem cells. Flow cytometry wasused to quantify infected cells (GFP-positive cells). Cells were platedto a density of 10⁴ and 24 hours later were infected with AdGFP orAdGFP-RGD at an MOI of 10. 72 hours after infection, cells were examinedfor green fluorescence by flow cytometry. Cultures infected with Ad-GFPshowed 13.5±5.4% positive cells and cultures infected with Ad-GFP-RGDshowed 30.1±7.2% positive cells. Low infectivity of mesenchymal stemcells with the virus of unmodified tropism is consistent with the lowexpression of CAR.

Delta-24-300 Replication in vivo. In order to determine if Delta-24-300replicates in vivo, a study was performed in two animals bearingintracranial U-87 MG xenografts (5×10⁵ cells inoculum) infected withDelta-24-300 or UV-inactivated Delta-24-300 (control) as a single doseof 10⁸ pfiu/tumor. Brain MRl was performed on Days 7 and 14 after cellimplantation and then sacrificed. The MRI scans, shown in FIG. 30,showed growth progression in the control tumors, but no growthprogression in the tumors infected with Delta-24-300. These results wereconsistent with histopathologic examination of the brains collected atsacrifice, which showed inclusion bodies around areas of necrosis in thetumors infected with Delta-24-300, suggesting the presence of newadenoviral progeny. There was no evidence of necrosis in the tumorsinfected with control UV-inactivated Delta-24-300 virus. The replicationproperty of the Delta-24-300 virus was confirmed by expression of latestructural genes (hexon). In addition, the inclusion bodies werepositive for adenoviral proteins. Together, these data indicate thatDelta-24-300 replicates in vivo and suggest that infection of tumorswith this agent may result in therapeutic effect.

Analysis of Infectivity of Subventricular Area. To assess infection inthe subventricular zone, a series of archived U-87 MG tumors wereanalyzed and included (A) 10 brains with tumors treated with Delta-24,(B) 5 brains with tumors treated with Delta-24-RGD, (C) 2 brains withtumors treated with Delta-300, and (D) 2 brains with tumors treated withDelta-24-300 as described above. Cases in which the tumor showed highlevel of viral infection throughout the tumor and inclusion bodies wereobserved near the edge of the tumor tissue were selected.Immunohistochemical analysis of viral proteins did not reveal anyadenoviral-positive cells in the subventricular zone in any of theexamined brains. Therefore, the inventors believe that there is nospreading of adenoviral infection from the tumor to the subventricularzone.

Human glioma cell lines To test the effect of D24-300 and D24-300vIII invitro, glioma cell lines have been selected that have 80-100%transduction efficiency with replication-competent adenoviral vectors(U-87 MG, U-251 MG, D-54 MG) (Fueyo et al., 1996a), and whereDelta-24-mediated anti-cancer effect has been already tested. The U-87MG cell line (ATCC) was developed by Nikshikawa et al. (1994). Briefly,U-87 MG cells have been stably transfected with wt-EGFR (U-87 MGwtEGFR)or mutant EGFR (U-87 MGΔEGFR), expressing wt EGFR or EGFRvIII,respectively.

Other cancer cell lines and normal human cell cultures. The followingcell lines from the ATCC will be used: Saos-2 and U-2 Os (humanosteogenic sarcomas); NCI-H446, NCI-H209 and NCI-H146 (human small cellcarcinoma of lung); PC-3 and DU141 (human prostate carcinoma);MDA-MB-157, MDA-MB-231, and MDA-MB-361 (breast carcinoma). Normal cellcultures from ATCC that will be tested for infectivity and for theeffect of the adenoviral construct include Normal Astrocytes(Clonetics), CDD-29Lu and CDD-33Lu (human lung fibroblasts); CDD-33Co,CDD-18Co, and CDD-1 12Co N (human colon fibroblasts), BUD-8 (human skinfibroblasts); HU-VEC-C (human endothelial cells). Human stem cultureswill be represented in the in vitro experiments by mesenchymal stemcells and neural progenitors cultures (Cambrex, Walkersville, Md.).Culture conditions will be performed as recommended by manufacturers.

Cell Synchronization. Cells will be serum-starved for 3 days byculturing in MCDB-105 serum-free medium (Sigma St. Louis, Mo.). Tostimulate synchronous cell cycling progression, medium will be replacedwith DMEM containing 10% fetal calf serum.

Infection Conditions. Infection of cell lines will be carried out bydilution of viral stock to particular concentrations, addition of viralsolutions to cell monolayers (0.5 mL per 60 mm dish) and incubation at37° C. for 30 min with brief agitation every 5 min. The infected cellswill be returned to the 37° C. incubator.

PCR Analysis. This test will be used to detect the presence of wild-typeadenoviruses, adenoviruses carrying wild-type E1A (Fueyo et al., 2000).

BrdU Analysis. Cells will be pulse-labeled with BrdU for 2 h beforecollection. After BrdU labeling, adherent and nonadherent cells will becombined, resuspended in 0.5 mL PBS, and fixed in 5 mL 70% ethanol, 50mM glycine (pH 2.0). Samples will be subjected to denaturation in 0.5 mL4M HCl for 20 min and then incubated at 37° C. for 1 h in 0.1 mL PBScontaining 0.5% BSA, 0.1% Tween and 20 μL FITC-conjugated anti-BrdUantibody (clone BMG 6H8, Roche Molecular Biochemicals, Indianapolis,Ind.). After incubation, samples will be centrifuged and resuspended ina 0.5 mL solution containing 500 μg/mL RNase A and 10 μg/mL propidiumiodide in PBS. Cells will be analyzed by two-color flow cytometry forBrdU incorporation and DNA content.

Immunofluorescence. Cells will be seeded at 10⁵ cells per well intwo-well chamber slides, allowed to grow for 20 hours and then infectedwith Delta-24-300. 24 hours later, cells will be fixed with methanol for4 minutes at −20° C. The slides will be incubated for 10 minutes withDAKO protein block serum-free and then incubated with primary antibodies(anti-E1A, Santa Cruz Inc., Santa Cruz, Calif.; anti-BrdU (Biogenics,San Ramon, Calif.). Cells will be pulse-labeled with BrdU for 2 hoursbefore collection. The slides will be washed twice for 5 min with PBSand fluorescent antibody (FICT and Rho) will be added. Slides will becovered with mounted media and fluorescence detected under a fluorescentmicroscope.

CPE Assays. As described above, see Fueyo et al., 2000.

Viral Replication. Viral production will be quantified by plaque-formingassay. 36 hours after infection, cells will be scraped into culturemedium and lysed by three cycles of freezing and thawing. Cell lysateswill be clarified by centrifugation and the supernatant will be seriallydiluted in medium for infection of 293 cells in 6-well dishes. After 1hour of incubation at 37° C., the infected cells will be overlaid with 3mL 1.25% SeaPlaque agarose (FMC, Rockland, Me.) in DMEM/F12 with 10%fetal bovine serum. Additional agarose will be added to each dish 4 dayslater. Plaques will be visualized 7 days after infection.

Immunohistochemistry. The presence of adenoviral E1A and hexon proteinsin the treated xenografts will be assessed by immunohistochemistry.Paraffin-embedded sections from tumors will be de-paraffinized andrehydrated through xylene and ethanol into PBS. Endogenous peroxidaseactivity will be quenched by incubation for 30 minutes in 0.3% H₂O₂ inmethanol. Sections will be treated with goat anti-hexon (Chemicon Inc.,Temecula, Calif.) or goat anti-E1A (Santa Cruz Inc., Santa Cruz,Calif.). Immunohistochemical staining will be performed according to themanufacturer's instructions with diaminobenzidine by using VectorLaboratories ABC kits (Amersham).

Selective Infectivity of the vIII-Adenoviral Constructs To ascertainwhether the insertion of the EGFRvIII-chimeric fiber motif isresponsible for selective infectivity, two different studies will beperformed with an established system where U-87 MG cells have beenstably transfected with either EGFRvIII or wild-type EGFR.

Infectivity analyses (GFP detection): Briefly, 48 hrs after infection,cells will be collected by trypsinization, washed with PBS, and stainedwith 50 mg/mL propidium iodide and 20 mg/mL RNAse for 15 min at roomtemperature. Samples will be analyzed with an EPICS XL-MCL flowcytometer (Beckman-Coulter, Inc., Miami, Fla.) by using a 488-nm argonlaser for excitation. Fluorescence will be detected through 520 bandpass filter. All cytometric data will be analyzed with the System IIsoftware (Beckman-Coulter, Inc.).

Competitive inhibition assay: PEPHC1 peptide and 6H (control) peptidewill be produced synthetically. Cells will be cultured in 96-wellplates. 20-24 hours later, PEPHC1 peptide (1 mg/ml) will be added to theculture. Ten minutes later, cultures will be treated withD24-300vIII-EGFP, D24-300-FF6H-EGFP, or D24-300-EGFP. 48 hours later, adirect examination of the cells with fluorescence microscopy will takeplace, followed by determination of the percentage of green cells(fluorescence) after scoring 500 cells using phase contrast.

Detection of EGFR or EGFR vIII (Immunohistochemistry): An anti-EGFRantibody (Cell Signaling, Beverly, Mass.) will be used that recognizesboth EGFR and EGFRvIII and an anti-EGFRvIII antibody (Zymed LaboratoriesInc., San Francisco, Calif.) that specifically recognizes EGFRvIII.

Intracranial implantation and treatment of the tumors. The implantableguide-screw method described above (Lal et al, 2000) will be used. PETscan will be used to determine the antiglioma efficiency of theoncolytic system targeting EGFRvIII.

Correlation of EGFRvIII expression with survival. The invnetors plan toperform in vivo studies with 3 cell lines expressing different levels ofEGFRvIII and wild-type EGFR: parental U-87 MG, U-87 MG-DEGFR (highexpression levels of EGFRvIII), and U-87 MG-wtEGFR (high expressionlevels of wild-type EGFR). The studies with the U-87 MG system (very lowCAR expression) address the question of the relevance of EGFRvIII on theanti-cancer effect in a system in which all other factors are similar inboth cell lines and can therefore be subtracted in the interpretation ofthe results.

Besides the study of viral replication inside the tumor, thehistopathological examination of the brains will allow the examinationof other interesting aspects involving adenoviral distribution in vivo.Viral particles will be detected by immunostaining for viral proteins ashexon and E1A, as well, as detection of viral inclusions. Because theproliferative potential of precursors cells, and thus, the possibilityof supporting viral replication, the subventricular zone, thesubgranular zone, the hippocampus and cortex (Gage, 2000) will beexamined systematically for the expression of adenoviral proteins.Double immunoflourescence for E1A/hexon and nestin will be performed toelucidate the existence of viral particles in precursor cells.

Engrafting Human Glioma Cells and Intratumoral Injections 5×10⁵ U-87 MGcells will be engrafted into the caudate nucleus of athymic mice, usinga guide-screw system as previously described (Lal et al., 2000). Theanimals showing general or local symptoms of toxicity will besacrificed. The surviving animals 100 days after engraftment. Brainswill be fixed in 4% formaldehyde for 24 h and then embedd them inparaffin. Animal studies will be performed in the veterinary facilitiesof M. D. Anderson Cancer Center in accordance with institutionalguidelines.

Heterotransplants Tumor specimens from tumor lines already establishedwill be obtained and will used to generate tumor lines.Heterotransplants will be minced and injected subcutaneously into theright flank of nude mice in a volume of less than 1.0 ml using a16-gauge needle. Treated and control tumors will be measured once ortwice weekly with calipers, and the volumes will be calibrated accordingto the formula a2Xb/2, were a=width and b=length. For tumor passage, theanimals will be euthanized, tumors will be removed under sterileconditions, and, after appropriate material will be obtained forhistological analysis, the tumors will be passed in a modified tissuepress through 30/40 mesh cytosieves. Volumes of 50-200 μl of thisprocessed tissue will then be implanted into the right flank of therecipient animals. Serially passed tumors will be followed until avolume of at least 1000 mm³ is achieved. Volume doubling times will becalculated from sequential measurements once exponential growth begins.A volume of 500 mm³ will be tacked as a measure of successful growth asvolumes will probably fluctuate somewhat below this level, andprogressive growth is expected to occur 500 mm³ will be on the linearportion of the growth curve in all instances. To asses tumor morphologyportions of subcutaneous lesions from all mice that die spontaneously orwill be killed will be fixed for at least 48 hours in 10% bufferedformalin, embedded in paraffin, cut into 5/7 micron sections, andstained with H&E. These slides will be coded and evaluated for thepresence of neoplastic cells.

B. Results

The Delta-24 oncolytic system will be modified to improve the ability ofthe adenovirus to identify cancer cells. In order to improve theselectivity of current gene therapy strategies, the inventors willtarget the oncolytic adenoviruses to specific cell-surface receptors.Compared to wild-type adenovirus, the proposed adenoviral constructs aredesigned to infect through a mutant form of EGFR, known as variant IIIor EGFRvIII (Kuan et al., 2001). This mutant receptor has been foundexpressed only in cancer cells, including approximately 30% ofglioblastoma multiforme, which is the most frequent and most common formof gliomas. EGFRvIII encompasses an in-frame deletion of exons 2 through7 (amino acid residues 6-273) in the extracellular domain. Due to thepresence of this deletion, peptides can be designed to bind specificallyto EGFRvIII, but not to any of the wild type forms of EGFR. Sincemalignant gliomas express low levels of adenoviral receptors,redirection of adenoviruses to cancer-related receptors, such asEGFRvIII, should result in a high therapeutic index through theimprovement of efficiency in the infection of cancer cells and inabilityof infection of normal cells.

The inventors will replace the wild-type fiber structure of the proteinwith a chimeric fiber that has been constructed using a commerciallyavailable T4 fibritin DNA (Krasnykh et al., 2001), a DNA linker, andfinally, the binding peptide. The peptide will be the “contact area”with the host cell. An adenovirus, which has been modified to express achimeric fiber to bind the EGFRvIII receptor, will be able to bind andinternalize exclusively into human glioma cells. In addition, anadenovirus directed to bind EGFRvIII should be able to infect humanglioma cells more efficiently than a wild-type adenovirus because gliomacells express low levels of the natural, main receptor for adenovirus(CAR). EGFRvIII was selected because it is one of the best-examinedsystems in gliomas. Also, at least one peptide has already beendescribed that is able to bind EGFRvIII. To test the ability ofDelta-24-300vIII and Delta-24-300 to infect cells exhibiting low CARexpression and differing levels of EGFRvIII expression, a system will beused that limits the differences between cells in regards to EGFRvIIIexpression. This system consists of a human glioma cell line, U-87 MG,which expresses low levels of CAR and EGFRvIII and has been geneticallymodified to constitutively express high levels of EGFRvIII (U-87MG-ΔEGFR) or EGFR (U-87 MGwtEGFR) (Nikshikawa et al. 1994). Comparisonsbetween the wild-type fiber and fiber-modified adenoviruses in thissystem should yield clear information about the infectivity capabilityof both constructs.

Targeting oncolytic adenovirus to either of EGFRvIII or EGFR shouldresult in adenovirus that preferentially infects cancer cells. These tworeceptors have been selected because both have been well characterizedand defined in human glioma cells. The urokinase plasminogen activatorreceptor, uPAR, protein has been targeted using peptides that allow theinternalization of adenoviral vectors (Drapkin et al., 2000). It hasbeen demonstrated that urokinase plasminogen activator, or a 7-residuepeptide derived from this protein (u7-peptide), binds the receptor andstimulates apical endocytosis. Both ligands enhanced gene transfer bynonspecifically binding adenovirus and adeno-associated viral vectors aswell as a modified adenoviral vector that was coupled to the u7-peptide.These data provide strong evidence that the uPA/uPAR system may offersignificant advantages for targeted oncolytic systems. The increasedexpression of certain fibroblast growth factor (FGF) family members,including basic and acidic FGF, has already been strongly associatedwith malignancy in human astrocytic tumors. Glioblastomas also expressan alternatively spliced form of FGFR1 containing twoimmunoglobulin-like disulfide loops (FGFR1 beta), whereas normal humanadult and fetal brain tissues express a form of the receptor containingthree immunoglobulin-like disulfide loops (FGFR1 alpha) (Yamaguchi etal., 1994). The selective expression of FGFR in malignant gliomas andthe presence of alternative spliced forms make FGFR (Jin et al., 2000) adesirable target for oncolytic adenoviral targeted anchorage in humanmalignant gliomas.

Selectivity and efficacy of glioma-targeted-Delta-24 in vivo. Theintracranial model of human glioma xenografts implanted in nude mice isone of the most representative procedures for the exploration of noveltherapies for brain tumors. The inventor will check the pathology of thetumors, the expression of viral proteins, and the spread of the virusthroughout the tumor at several time points in every experiment. Thestudy is designed to ascertain whether or not there is a correlationbetween the kinetics of the viral spread, the tumor suppression and theimprovement of survival. Survival data and pathological observationssupport the hypothesis that one can efficiently examine theadenovirally-mediated anti-cancer effect in vivo. Furthermore, thesedata are consistent with the prediction that tropism-modifiedadenoviruses are more powerful than wild-type fiber adenoviruses, suchas Delta 24, in a cell line expressing low levels of CAR.

The inventors will perform studies with two different animal models.First, the inventors shall use the U-87 MG system. Glioma cells will beinjected intracranially into nude mice. In the second study, theinventors shall use heterotransplants of human gliomas expressing EGFRor EGFRvIII implanted subcutaneously in nude mice. This model isrequired because the expression of EGFRvIII is generally lost in humanglioma cell lines (Bigner et al. 1990). In addition, data from thismodel will complement those obtained from the U-87 MG model which isboth more accurate to analyze the dependence on the expression ofEGFRvIII for adenoviral infection, but more artificial than theheterotransplant system. The U-87 system and the heterotransplants aretwo complementary models in the way that in the U-87 MG system, allcells have a similar genetic makeup with the exception of the artificialexpression of EGFR or EGFRvIII. In the heterotransplant model, thecommon characteristic between all the tumors should be the abnormalexpression of EGFR or EGFRvIII with most probably different geneticabnormalities. The cell line model will be used to examine in detailadenoviral mediated anti-cancer effect. The second model will be used todetermine the specificity in a more realistic setting closer to theclinical scenario. Heterotransplants are also useful to examine thedegree of homogeneity in the ability of the targeted adenovirus toinfect tumors with different degrees of EGFRvIII expression.

Example 9 TIE2 Expression in Gliomas and its Involvment in TumorFormation

To examine the relationship of Tie2 expression in gliomas and thepossibility of Tie2 of being involved in tumor formation, the inventorsare applying two different and complementary approaches. One approachconsists of the isolation of Tie2+ cells from established human gliomacultures, and then characterize these populations for tumor stem-celllike and/or tumorigenic properties. A second approach is based on theisolation of tumor spheroids (stem-like cultures) from human gliomaspecimens, and subsequent analysis of the expression of Tie2, and thetumorigenic properties of Tie2+ populations in these tumorneuro-spheroids. Alternatively, the information can be obtained from thestable cell lines.

Functional characterization of the Tie2-positive tumor cell populationand its significance in glioma tumor formation in vivo. The inventorshave collected convincing data on the expression of the tyrosinereceptor Tie2 in human glioma cells in culture, in gliomaculture-derived intracranial xenograft, and importantly in malignantglioma human specimens. Tie2 expression was not detectable in humannormal brain. Tie2 transcript was present in tumor neurospheres derivedfrom human glioma tumors in co-existence with CD133 (stem cell marker).Furthermore, a small population of partially differentiated neuralprecursors was positive for Tie2 expression. To determine the role ofTie2 in gliomas, A172 glioma cell line was divided into two populations:Tie2+ and Tie2−.

Expression of Tie2 in human malignant gliomas is shown in FIG. 18.RT-PCR analysis was performed on mRNA extracted from human glioma celllines and cultures. Primers and conditions for the PCR reaction werepublished previously (Poncet et al., 2003). A 503-bp fragmentamplification was obtained from RNA extracted from the majority of thecell lines and high Tie2 RNA levels were present in A172, U-87 MG, andD54 MG. Sequencing of the amplified product in these cell linesconfirmed the presence of the Tie2 transcript. Futhermore, Tie2expression was detected by western blotting using the membranesubfraction lysates from different cell lines. Anti-human Tie2 antibodyrecognized a 140-kDa band in the membrane proteins subfraction ofHU-VEC-C, U-87 MG and D-54 MG, Al 72, but not in U-251 MG.

Cytosol fraction proteins were negative for Tie2 expression. Becauseexpression of RTKs have been reported being different in vitro than invivo, U-87 MG cells were implanted in the brain of nude mice and Tie2expression was assessed in sections of those xenografts. Tie2 levelswere analyzed using two different antibodies (Santa Cruz, R&D), andcompetitive inhibition of the epitope/antibody reaction, was obtainedwith Tie2 peptide. Tie2 expression was in the endothelial cells of sometumoral vessels and peritumoral vessels, as well as in the gliomacompartment. To determine whether the expression of Tie2 in human gliomacell lines was an artifact originated by clonal selection or in vitroculture, a series of human gliomas and normal brain tissues wereexamined using a microtissue array (Wang et al., 2004). Theseexperiments showed that Tie2 is frequently expressed in Grade III andGrade IV gliomas, but is not expressed in normal brain. As expected,Tie2 was present in most tumoral vessels in every tumor grade.

Data shows that Tie2 is present in primary tumor neurosphere culture.Cultures were established from 3 GBMs tumors, which were acutelydissociated into individual cells. Culture conditions were used thatfavored stem cell growth, established previously for isolation of neuralstem cells as neurospheres (Galli et al., 2004). SFM allows for themaintenance of an undifferentiated stem cell state, and the addition ofbFGF and EGF induced the proliferation of multipotent, self-renewing,and expandable neural stem cells (Reynolds et al., 1996). Within 7-14days of primary culture a subset of the GBMs tumors (3 out of 5) yieldeda minority fraction of cells that demonstrated growth intoneurosphere-like clusters, or tumor neurospheres. Analysis of primaryand secondary tumor neurospheres generated from the three specimensshowed expression of nestin transcript (an intermediate filament proteinfound in undifferentiated central nervous system cells and acharacteristic neural stem cell marker), CD133 transcript, a novelputative neural stem cell marker (Singh et al., 2003 and 2004), andBmi1, a molecule necessary for neural stem cell renewal and early neuralprogenitors (Valk-Lingbeek et al., 2004). The tumor spheres did expressTie2 (please note that tumor should contain glial and endothelial Tie2).Furthermore, RT-PCR was used to detect the presence of CD133 and nestintranscript in A172 and U-87 MG glioma cells lines (Tie2+ cancer cells).As control, cultures of cortical mouse astrocytes were tested (McCarthyet al., 1980), which were negative for CD133, and Tie2, but positive forGFAP, showing that Tie2 expression is not the result of tissueculture-originated artifact. Glioma sphere have been tested forself-renewal activity by the ability to form secondary and tertiaryspheres. The morphology and expression of several stem-like cell markersdid not varied or increased in those consecutive populations, showingpositive expression of Tie2, CD133, and Brm1.

Next, conditions used for normal neurosphere differentiation wereapplied to primary tumor spheres to determine whether Tie2 expressionwas present in partially differentiated cells. After differentiationwith 10% FBS for 1 day, immunocytochemistry was performed on tumor stemcells using GFAP (for astrocytes), and Tie2 antibodies. Strikingly,dissociated tumor spheres from the specimen tested grown adherently andin serum expressed GFAP, recapitulating the astrocytic lineage of thetumor. Interestingly, Tie2 expression was present in some of thepartially differentiated cells.

Tie2 + populations were isolated from A172 human glioma cell line. Tobetter define the significance of Tie2 expression on glioma cells, theexpression of Tie2 was analyzed using flow cytometry, and Tie2 positiveand negative cell populations were sorted. Flow cytometricquantification of Tie2 expression in A172 glioma cultures was 11.2%.When tumor cell cultures were sorted for Tie2 expression, Tie2 positive(1.2% total population) and negative (16.5%) cell populations werecollected and cultured separately in serum-free neural stem cell medium.Tie2− and Tie2+ A172 cells were cultured in suspension in this media.Although some cells died in this culture condition, approximately 50% ofthe Tie2+ cells remained viable showing with morphological signs ofactive mitosis, at the moment of that submission.

Growth kinetics in a human glioma intracranial animal model. Afterintracranial injection of 5×10⁵ U-87 MG cells into the right basalganglia of nude mice (day 0), the tumors grew from 0.02 mm³ on day 4 to100 mm³ by day 20. All animals died by day 30. Serial temporalexamination of the brains of tumor-bearing animals showed centralnecrosis of the xenografts within 4 days. At that time, the vesselssurrounding the tumor displayed changes in morphology, including anenlarged diameter and a disorganized structure. After day 4, necrosiswas not observed. From days 15 to 20, the tumors were large(41.2±6.3mm³) and hypervascularized with large vessels (36.7±3.4 and53.1±12.9 vessels/0.5 mm²). After day 20 and (104.66±7.1 mm³) thevessels were numerous (53.1±12.9 vessels/0.5 mm²). Staining forproliferating cell nuclear antigen revealed a high proliferativeactivity ranging from a few hours after implantation (>80% cells).

Characterization of the Tie2 transduction signaling in glioma cells andits impact in the glioma phenotype in vitro. The invnetors havegenerated data in support of a connection between the Ang1/Tie2 pathwayand the Ras/p42/p44 MAPK signaling pathway. In addition, modulation ofTie2 signaling seems to be related to proliferation, adhesion andmigration of human glioma cells. Of importance, Ang-2-mediated blockadeof this pathway blockade results in a prolonged survival of U87MG-bearing animals.

To explore the regulation of Tie2 signaling by Angs in U-87 MG cells,Tie2 was immunoprecipitated from membrane fractions of U-87 MG cellstreated with rAng1 (500 ng/ml) for 10 min, and phosphorylation of Tie2was addressed using anti-phosphotyrosine antibody. Interestingly,treatment of Ang1 induced phosphorylation of Tie2 in U-87 MG cells,suggesting that Tie2 receptor is functional in U-87 MG cells. Therefore,the antagonizing effects of rAng-2 on Ang1-induced signaling pathway inU-87 MG cells was investigated. Inhibition of Tie2 phosphorylation byAng1 treatment in rAng-2 treated U-87 MG cells was observed.

The Ang-2/Ang1/Tie2 system has been studied in endothelial cells, anddifferent pathways have been reported as involved in the cascadesignaling. The inventors have obtained results that suggest that Ang1induces MEK/ERK stimulation in glioma cells. MAPK activity was measuredby the degree of phosphorylation of two MAPKs, ERK1 (p44^(MAPK)) andERK2 (p42^(MAPK)). Consistant with previous reports (Kim et al., 2002;Toumaire et al., 2004), rhAng1 (100 ng/ml) increased ERK1/2phosphorylation of HU-VEC-C endothelial cells. Of interest, treatment ofU-87 MG with rhAng1 also increased ERK1/2 phosphorylation. Co-treatmentwith Ad-transduced Ang-2 inhibited the Ang1 induced ERK1/2phosphorylation. Of importance for establishing a definitive linkbetween Tie2 and MAPKs, the presence of soluble Tie2, partiallyinhibited ERK1/2 phosphorylation.

In addition, the inventors have obtained data regarding the upstreameffector of p42/p44 MAPK, Ras, by analyzing Ras activity inAng1-estimulated U-87 MG cells. Ras activity, which detects activeGTP-bound Ras, was performed by immunoprecipitation of Ras-Raf complexes(contains only active Ras), and consequent western blotting detection ofRas-GTP molecule. The inventors results show that Ras activity increasesafter Ang1-stimulation, suggesting the idea of Ang1/Tie2 signallingthrough Ras/MAPK in gliomas.

Consistant with previous reports, Ang-1/Tie signaling in endothelialcells in vitro involves PI3K/Akt activation (DeBusk et al., 2004;Papapetropoulos et al., 2000). Similar experiments were performed by theinvnetors confirming the Ang1 effect on Akt phosphorylation in HUVEC(endothelial cells); however, treatment of U-87 MG glioma cells withrhAng1 (100-500 ng/ml) did not modify the pattern of phosphorylation ofthe Akt molecule.

Tie2 can also recruit additional signaling molecules that participate incellular pathways that affect the shape and migratory properties ofcells. In this regard, the Tie2-associated docking protein Dok-R canpotentiate NCK-dependent endothelial cell migration in response to Ang1(Master et al., 2001; Jones et al., 2003). Our preliminary resultsperformed in stable transfected Tie2-U-251 MG showed that stimulation ofTie2 with Ang1 results in Tie2 association with Dok-R protein.

In order to generate a cell system suitable for the analyses of theTie2's effect in cell growth, cell invasion, and tumorigenicity, theinventors have constructed an isogenic U-251 MG that constitutivelyexpresses Tie-2. The generation of the cell line involved the stabletransfection of Tie-2 cDNA (Audero et al., 2004). Transfected cells havebeen characterized by immunoblotting for basal and p-Tie2 expression, aswell as flow cytometry studies.

In addition and as a complementary system, the invnetors planned tomodify the Tie2-positive U-87 MG (ATCC). For that purpose, Tie2 siRNAhas been used (Santa Cruz Biotechnology) (Niu et al., 2004). Since bothU-251 MG and U-87 MG express a mutant PTEN and therefore arecharacterized by high levels of p-AKT, a LNN29 isogenic cell lineexpressing Tie2 will be generated. This cell line will be used toexamine the effect of the Tie-2 pathway in a glioma cell line expressinga wild type PTEN protein.

Modulation of glioma phenotype by Angs/Tie2. Modulation of Tie2signaling was investigated in relation to the proliferative and survivalsignals on U-87 MG glioma cells. First, the viability of sustainedAng-2-treated U-87 MG cells were analyzed. Cell growth followed by 7 dindicated a decreased in viability of 20% of the culture (P<0.001), witha representation of 10% of apoptotic cells by TUNNEL assay (data noshown).

It was reported that Ang1 promotes adhesion of Tie2+ hematopoietic stemcells to fibronectin and collagen and also promotes the interaction ofendothelial cells with surrounding mesenchymal cells and theextracellular matrix (Davis et al., 1996; Suri et al., 1996; Arai etal., 2004). Therefore, the inventors analyzed the effects of Ang1/Tie2signalling on glioma adhesion. Briefly, 96-well dishes were coated for 2hr at room temperature (RT) with rAng1 diluted in PBS. Wells were thenblocked for 30 min at RT with 0.5% heat-inactivated BSA in PBS (80° C.for 10 min) and washed three times with PBS before adding cells. U-87 MGcells were harvested and resuspended. 30,000 cells were seeded/well, andthe plate was incubated at 37° C. for 1 hr. Nonadherent cells wereremoved and attached cells were fixed in 4% paraformaldehyde and stainedwith 0.2% crystal violet. Cells were solubilized in DMSO and absorbanceof each well was read at 570 nm. The data suggest that Ang1/Tie2 pathwayfunctions increasing adhesion properties of U-87 glioma cells (P<0.0001,double sided t-test). Moreover, addition of sTie2-Fc, in this culture,decreased the proportion of U-87 cells adhering to the Ang1-coated well,probably by blocking Ang1/Tie binding (P<0.003 vs. Ang1-treatedcultures, double sided t-test). Similar results were obtained when Tie2−and Tie2+ U-251 MG were used. In addition, the possibility ofAng1-induced adhesion has been assessed. For that study, the wells werecoated with several ECM and then, U-87 MG cells that were previouslystimulated with Ang1 (BSA control), were plated on those wells. Anincreased in the adhesion capabilities of Ang1-stimulated U-87 MG cellsto collagen I, fibronectin (both, P<0.0004), collagen IV and vitronectin(both, P<0.01) has been observed. No modifications were observed withlaminin or BSA-coated dishes (both, P>0.1), suggesting a indirect rolefor Tie2 in that experiment, what could be consistent with the reportedTie2-mediated upregulation of integrins.

Chemotactic Migration. Witzenbichler et al. (1998) have reported thechemotactic properties of Ang1 for Tie2-endothelial cells. A chemotaxisassay was performed in the presence or absence of Ang1. Incubation withvarious concentrations of Ang1 enhanced chemotactic migration of U-87 MGcells. Migration assay was performed as described previously(Gomez-Manzano, 2003). Briefly, the lower surface of the filter wascoated with gelatin. 600 μl of DMEM/F-12 (1:1) containing rAng1 and 0.1%BSA was added into lower compartment of the Transwell chamber, and 100μl of cell suspension was added in the upper chamber. After 24 h ofincubation, the filters were fixed, stained with H/E, and the cells onthe lower surface of the filters were counted at 400× with Zeissmicroscope. Assays were performed in triplicate. Ang1 led to asignificant increase in directed migration of U-87 MG cells (P=0.028,double-sided t-test). To distinguish between chemotactic andchemokinetics effect of Ang1 on U-87 MG, an analysis was performed wheresimilar doses of Ang1 were present above and below the filter.Significant migration was observed only when a concentration gradient ofAng1 was established, a finding typical of factors inducing chemotaxis.In contrast, adding equivalent concentrations of Ang1 to both sides ofthe filter did not enhance cell movement (P=0.28), thereby excluding asignificant chemokinetic effect of Ang1. Counterpart experiments usingsimilar doses of Ang-2, showed that Ang-2 is not a chemotactic for U-87MG human glioma cells.

Effect of Ang-2 expression on glioma tumorigenicity. Overexpression ofAng-2 modifies intracranial tumorigenicity properties in the U-87 MGcell line. In this study, the U-87 MG cell line was infected withAdAng-2 or an adenovirus control, AdCMV, and 3 days later, cells wereimplanted intracranially in nude mice. These experiments showed that allcontrol-treated U-87 MG cells (n=17) developed into intracranial tumorsthat ultimately caused the death of the animals by day 38. The tumorswere ellipsoid masses that compressed anatomical structures in theipsilateral and contralateral hemispheres of the brain and were similarto those formed in other experiments using U-87 MG cells (Fueyo et al.,2003). Conversely, animals bearing intracranial Ang-2-treated U-87 MGcells had a longer survival (P<0.0001, log-rank test); and importantly,3 of them (3/17; in controls: 0/17) longer than 100 days without signsof neurological disease. These studies showed sustained expression ofAng-2 lead to significant increased on survival. Examination of thebrains of long-term survivor animals did not revealed residual tumor.Time point analyses were performed were animals were sacrified 36 h and10 days after cell implantation. Analyses of Ang-2-U-87 MG derivedtumors within 36hrs of implantation showed enhanced expression of Ang-2.10d after cell implantation, Ang-2-U-87 MG derived tumors showed bigareas of necrosis and fibrinoid necrosis of tumoral vessels. Thesetumorigenicity studies strongly show that overexpression of Ang-2resulted in inhibition of tumor production or markedly interference withthe progression of tumor growth, and suggest a relationship between Tie2signalling and glioma development.

NLLMAAS (SEQ ID NO:20) peptide specifically interacts with Tie2. In anattempt to identify peptides that specifically interact with and blockthe Tie2 pathway, Toumaire et al. (2004) screened a phage-displayedpeptide on a recombinant Tie2 receptor. One peptide, NLLMAAS (T4),completely abolished by binding to the Tie2 receptor, the binding ofboth Ang1 and Ang-2. Testing the binding of the proposed Tie2-ligand,T4, the inventors synthesized the peptide linked to FAM signal that wassuitable for flow cytometric detection. Peptide was synthesized byautomatic solid phase chemistry using Fmoc strategy. The N-terminalaminohexanoic acid was labeled by coupling 5(6)-carboxyfluorescein.Peptides were purified to >98% purity by reverse phase HPLC. Theinventors have obtained data confirming by flow cytometric methods, thatthe peptide T4 binds preferentially to the membrane of cellsoverexpressing Tie2, using U-251 MG isogenic system, and A172.

Tie2+ populations derived from cell lines. The inventors have isolatedTie2+ and Tie2− populations from A172 human glioma cell line by flowcytometric sortening. Similarly, Tie2+ and Tie2− populations will beisolated from U-87 MG cell line. Sorted Tie2+ and Tie2− aliquots fromeach cell line will be examined by flow cytometry to evaluate theefficiency of sorting and purity of both populations will be analyzed.Parallel studies will be performed by immunohistochemistry analysis ofTie2 expression.

Tie2 expression in tumor spheres derived from human glioma tumors.Cultures have been established from 3 glioblastoma multiforme tumors,which were acutely dissociated into individual cells, and cultured. Eachbrain tumor yielded a minority fraction of cells that demonstratedgrowth into neurosphere-like clusters, termed tumor spheres. These tumorneurospheres showed some properties related with stem cell populations,as self-renewal (formation of secondary and tertiary neurospheres) andwere positive for stem-cell markers. Of interest, Tie2 expression waspositive in these cultures.

Frequency of stem cells populations within every tumor will bedetermined by primary sphere formation assay. Limited dilutions will beperformed as described previously (Singh et al., 2003). Spheres will bedissociated and plated in 96-well microwell plates in 0.2 ml volume ofserum-stem cell line medium. Cultures will be fed 0.025 ml of this mediaevery 2 days until day 7, when the percentages of wells not containingspheres for each plating density will be calculated and plotted againstthe number of cells per well. For primary sphere formation assays, thisanalysis will be performed on the entire acutely dissociated tumor cellpopulation on day 0 to quantify stem cell frequency within the tumor.

Purity of brain tumor stem cell population. Because normal stem cellscan migrate to sites of injury, and brain tumor cultures may potentiallybe contaminated with some normal neural stem cell, a conventionalcytogenetic analysis will be conducted to demonstrate that the gliomatumor stem cells being isolated are indeed transformed and are notnormal brain cells.

Identification of double Tie2/CD133 population. The co-existance ofTie2+ and CD133+ cells will be analyzed by using double immunostainingand flow cytometric analysis to detect these markers in the membrane ofthe isolated population from both, Tie2+ cells from glioma cellcultures, and tumor neurospheres from GBMs specimens.

Study of the potential of Tie2+ cell populations for self-renewal andproliferation. Neurosphere-initiating cells will be assessed forself-renewal activity by examining the replating activity of singleviable cells from the Tie2+-sorted/expanded neurosphere cells. Cellsderived from neurophere cultures (single neurospheres in 96-well dishes)initiated from Tie2+-sorted cells should consistently reinitiate theformation of secondary neurospheres. The morphology of secondary tumorspheres and the maintenance of expression of the neural stem cellmarkers nestin, CD133, Brml, will be assessed, as well as maintenance ofTie2 expression. Proliferation ability of Tie2+cells will be assessed byplating 1000 cells/well, and quanifying the number of viable cells ondays 0, 3, 5, and 7 after plating using a colometric assay. In addition,MIB-1 index will be analyzed for the tumor neuropheres.

Tumorigenicity in the initial tumor. Tie2 expression in human gliomacell lines: correlation with tumor formation. The main question for thisexperiment is the importance of Tie2+ populations in tumorigenesis orglioma initiation. A time point analysis of the evolution of thehistology and growth patterns of intracranial U-87 MG-derived xenograftshas been performed. Tie2+ and Tie2− U-87 MG cells will be injectedintracranially in nude mice. Two weeks after cell implantation animalswill be sacrified and brains collected and analyzed for incorporation oftransplanted cells into the brain. Analysis will be based on theformation of tumor. In the case of tumor formation the inventors willexamine if tumors maintain similar characteristics of U-87 MGparental-derived tumors, such as vascular proliferation (factor VIII,CD31), MIB index, and/or Tie2 expression.

Statistical methods. Fisher's exact test will be used to assessdifferences between the Tie2+ and the Tie2− groups. The inventorscontemplate that at least 90% of the Tie2+ mice will have tumor 2 weekspost intra-cranial injection while no more than 20% of the mice in theTie2− group will have tumor at this time.

To determine if there is a dose-response relationship within the Tie2−and Tie2+ cell lines a Bayesian generalized linear model will be used toassess the dose response relationship for the Tie2− and Tie2+ celllines. In this study mice will be randomized to a combination of one ofthree cell concentrations and also to Tie2− or Tie2+ cell lines. Notethat the cell lines are derived from specific patient's tumors thus theinventors have included a cell line source random effect in this model.The response variable for this model is the presence or absence of tumorwithin a given mouse. This methodology models association between pairsof responses for a given patient with log odds ratios.

The inventors contemplate using 2 mice per treatment group/cellline/cell source combination (total mice=2 mice×6 trt grps/Cell linecombinations×10 cell sources=120 mice). With a total of 20 micereceiving 10³ Tie2− cells and 20 mice receiving 10⁵ Tie2-cells, theFisher's Exact test will have 97% power to detect a difference of 70%tumorigenesis rate in the 10⁵ Tie2− cells group vs. a 10% tumorigenesisrate in the 10³ Tie2− cells group.

To analyze the survival data, survival curves will be estimated usingthe Kaplan-Meier method. The inventors will use Cox proportional hazardsregression analysis to estimate the hazard ratio between groups alongwith a 95% confidence interval for this ratio and a likelihood ratiop-value for testing if the ratio is different from 1 (the value of theratio if the groups have the same survival distributions). The hazardratio quantifies the relative rates of death between the groups. Basedon historic data the inventors expect the control animals to have amedian survival of 20 days.

A second group of experiments will be focused in analyze the anticancereffect with viral spread (Fueyo et al., 2003). The inventors willperform similar studies as above, however animals will be sacrified atdifferent time points after cell implantation: 10 and 20 days, andbrains will be extracted and analyzed for: (a) tumor size, (b) areas ofnecrosis, (c) viral spread, and (d) localization of the virus aroundnecrosis/microcystic areas, and compare with U-87 parental-derivedtumors. The study will reflect adenoviral infection and replicationwithin tumor cells.

For each measure, Spearman rank correlation analysis, which is sensitiveto general monotonic relationships and is robust to outliers in thedata, will be performed.

Tie2+ population in tumor-stem cell like populations. To determine ifthe Tie2+ populations from human glioma specimens are related to thehuman brain tumor initiating cells, the inventors will transplant Tie2+sorted/expanded neurosphere cells into nude mice. Briefly, expandedTie2+ and Tie2− sorted neurosphere cells at passage 6-10 will beharvested and gently dissociated with collagenase. 10³, 10⁴, 10⁵ cellswill be transplanted from every group (Tie2+ and Tie2−) into the brainof nude mice. Three weeks after cell implantation animals will besacrified and brains collected and analyzed for incorporation oftransplanted cells into the brain. Analysis will be based on theformation of tumor. In the case of tumor formation all the key WHOdefining features of GBMs will be assessed by H&E staining of theglioblastoma xenograft (mitotic figures, vascular proliferation, nuclearatypia, and pseudopallisading necrosis). In addition, the in situexpression of neuronal (β-tubuline 3) and glial (GFAP) cells will beexamined, as well as precursor markers (Nestin, CD133), and Tie2expression. The results will be compared to the original sample from thetumor (cryostat section from original tumor). Finally, proliferationproperties by expression analysis of Ki-67, will be quantify andcompared to the original tumor.

Modulation of Tie2 pathway. Modulation of Tie2 pathway will be studiedusing two different approaches. First, acute stimulation usingrecombinant protein Ang1 (rAng1) will be studied using time-dependentand dose-dependent studies. Counterpart experiments will consist oncompetitive inhibition by treatment with rAng-2, soluble Tie2 receptor(Tie2-Fc), or T4 peptide (as Toumaire et al., 2004).

Second, effect of sustained Tie2 stimulation will be also studied, as itis a more similar to the in vivo scenario, where Ang1 and Ang-2 aresecreted continously in the tumor environment. For that purposeadenoviral vectors expressing Ang1 will be used (Genetech, Calif.), andcompetitive inhibition will be performed by the use of an adenovirusexpressing Ang-2.

Development of a differential Tie2 expression system. The inventors havetested the expression of endogenous Tie2 in a panel of glioma cell linescell lines. Two Tie2-positive glioma cell lines, U-87 MG, D54 MG, andtwo Tie2-negative glioma cell lines, U-251 MG and LN229 have beenchoosen for further study. The inventors have generated an isogenicsystem consisting of parental U-251 MG and Tie2-expressing U251.Similarly, U-87 MG cell line has been cloned and several clones havebeen isolated that differ in the levels of Tie2 transcript expression.The inventors have demonstrated that siRNA-Tie2 can inhibited more than80% of the Tie2 transcript. This strategy will be extended to theTie2-positive cell line D54 MG and the Tie2-negative cell line LN229.

Ang1/Tie-2 signaling pathway in glioma cells. Ang1/Tie2 system inendothelial cells has been related with migration, sprouting, andsurvival. In these cell lines, several groups have demonstrated thatthese processes are mediated through pathways that include PI3K/Akt,FAK, Raf/Ras/MAPK, and Dok-R/Dok-2/Nck/Pak. Although the significance ofAng1 and Tie2 in vasculogenesis is well established, the signaltransduction cascades initiated by the binding of Ang1 to the Tie2receptor have not been completely characterized. In addition, thetransduction signal trigger by Ang1-mediated activation of Tie2 isunknown in glioma cells. The inventors have found that Ang1/Tie2 isinvolved in MAPK activation, and no Akt activation, in U-87 MG cellline. In addition, active Tie2 recruits Dok-R in glioma cells, leadingto the hypothesis of Tie2 regulating glioma migration.

The role of Ang1/Tie2 in the activation of the Ras/MAPK pathway. Ang1had been previously shown to activate the MAPK signaling cascade inHUVECs. The inventors have data suggesting the presence of an activeTie2/Ras/MAPK pathway in U-87 MG glioma cells. Studies will be performedusing a panel of glioma cells, including Tie2± isogenic system. Forthese studies the inventors will determine whether Ang1-trigerredp42/p44 phosphorylation can be completely abolished by recombinantsoluble Tie2 receptor (sTie2), by the presence or absence of a series ofconcentrations of the Tie2-binding peptide T4, and by Ang-2. Theinventors will also study the role of Ras, an intermediary signalingmediator between receptors and ERK1/2. Ras-Raf complexes (containiningonly active Ras) will be immunoprecipitated, and consequent westernblotting detection of Ras-GTP molecule. The inventors have shown thatRas activity increases after Ang1-stimulation, suggesting that Ang1/Tie2signaling through Ras/MAPK in gliomas. Studies will involvepre-treatments with Tie2 blockers, as well as inhibitors of Ras (FTIs)and MAPK (PD98059) to ascertain whether there is a concatenation ofsignaling from Ang1/Tie2 to Ras/MAPK.

The role of Ang1/Tie2 in the activation of the PI3k/AKT pathway. Arecent study indicates that Tie2 activates PI 3′-kinase through anassociation with the p85 regulatory unit (Jones et al., 1999; Kontos etal., 1998). Although these findings have been observed without Ang1stimulation, this result suggests that Ang1 can activate PI 3′-kinasethrough Tie2 binding. Data produced by the inventors suggest thatAng1/Tie2 does not regulate PI 3/AKT activation in U-87 MG human gliomacells. Studies will be directed to ascertain whether Ang1 inducedphosphorylation of the Tie2 receptor results in the activation of thep85 subunit of PI 3′-kinase and increased the activity of PI 3′-kinasein the above isogenic cell lines. Cells will be seeded and grown for 24hours. Then, the medium will be change to medium containing wortmannin.Two hours later, rAng1 will be added to the cells at the indicatedamounts, and the cells will be incubated for the indicated times. Then aphosphorylation assay of the Tie2 or p85 subunit of PI 3′-kinase will beperformed with anti-Tie2 antibody (Santa Cruz Biotechnology) or anti-p85subunit of PI 3′-kinase (Upstate Biotechnology, Inc) according to themethod described by Maisonpierre and collaborators (1997) and Hu andcoworkers (1996). In addition, the phosphorylation of AKT will beassessed comparing basal and phosphorylated levels. If PI3K/Akt pathwayis activated in glioma cell lines upon Tie2 activation, complementarystudies to confirm the cellular responses to that pathway will involveblockade of that signaling by LY294002, and PTEN expression(Gomez-Manzano et al., 2003).

The role of Ang1/Tie2 in the activation of the Dok-R pathway. Additionof PI 3′ kinase inhibitors in cell motility assays blocksAng1-stimulated migration of both endothelial and nonendothelial cellsexpressing Tie2 as well as Ang-2-stimulated chemotaxis of endothelialcells. Interestingly, however, inhibition of PI 3′ kinase activity canonly partially inhibit the chemotactic effect of Ang1 on endothelialcells (Jones et al., 1999; Fujikawa et al., 1999), thereby implying thatadditional Tie2 binding partners may also contribute to Ang1-mediatedendothelial cell migration. Phosphorylation of Tie2 further results inits association with a docking protein related to downstream of kinaseDok-R (Jones and Dumont, 1998). The inventors will perform studies toascertain whether the Ang1/Tie2 signaling in human glioma cells involvesthe activation of Dok-R. The most straightforward approach is theperformance of Tie2 and DokR co-immunoprecipitation in the presence ofAng1 or Tie2-blocking agents in isogenic cell lines and in A172 humanglioma cell line (Tie2+). Recruitment of Dok-R to the activated Tie2receptor via its PTB domain results in the phosphorylation of Dok-R onmultiple tyrosine residues. Dok-R will be immunoprecipitated after Ang1stimulation followed by immunoblotting the precipitates forphosphotyrosine to establish whether Ang1-mediated activation of Tie2results in the phosphorylation of Dok-R. Phosphorylation of DokRestablishes binding sites for the Ras GTPase-activating protein and theadaptor protein Nck, both of which have been implicated in cell motilityand actin rearrangement (Jones et al., 2003). Coimmunoprecipitationstudies will then be performed to ascertain whether Ang1 triggeringinduces significant association of Dok-1 with PAK and Nck. To ascertainthat Ang1/Tie2 stimulates the activation of PAK kinase in glioma cells,the inventors will stimulate Tie2 positive glioma cells with mock, Ang1or EGF, and PAK immunoprecipitates will be subjected to an in vitrokinase assay to measure the ability of PAK to phosphorylate MBP (myelinbasic protein). If the experiments show that Ang1 stimulates PAKactivity, relationship between PAK and migration can easily tested byoverexpressing PAK and then, analyzing migration abilities of gliomacells.

Cellular responses elicited by activation of the Ang1/Tie2 pathway.Studies will be performed to analyze the impact of Ang1/Tie12 signalingin the glioma phenotype. For specificity purpose sTie2, T4 peptide, andAng-2, will be used, as well as integrin-blocking agents (RGD; see Fueyoet al., 2003). Signaling pathways will be modulated by specificinhibitors (wortmannin or LY294002 for PI3K pathway; PD98059 for MAPKactivity) to correlate signal pathways with cellular responses.

Survival. Although Ang1 does not stimulate the proliferation ofendothelial cells, it promotes survival of those cells (Koblizek et al.,1998; Kim et al., 2000). The inventors have shown that blockade of Tie2by sustained expression of Ang-2, leads to a decrease of viability inU-87 MG cell line. Studies will be performed using isogenic cell linesexposed to Ang-2, sTie2 and T4, with or without Ang1 treatment.Population doubling time, cell cycle analysis, and BrdU incorporationwill be assessed for the Ang1-treated isogenic systems and thencompared. Involvement of signaling pathways will be studied by analysisof Tie2 phosphorylation, activation of MEK or Akt (western blotting),and the use of specific inhibitors.

Colony forming assay. The effect of Ang-1 on colony formation abilityunder anchorage independent conditions in the above glioma isogenic celllines will be assessed. The inventors contemplate an enhanced ability toform colonies in the isogenic cell lines expressing Tie2 and a reductionin the number and the size of the colonies in cells treated with solubleTie2-Fc, Ang-2. It is also contemplated that exogenous Ang-I willsupport colony formation in a dose-dependent manner.

Tie2/Ang1 pathway in adhesion. In vitro experiments have shown that Ang1has little effect on proliferation, but that it potently stimulatesendothelial cell adhesion (Carlson et al., 2001; Xu et al., 2001). Itwas reported that Ang1 promotes adhesion of Tie2+ hematopoeitic stemcells to fibronectin and collagen (Sato et al., 1998; Takakura et al.,1998), and also promotes the interaction of endothelial cells withsurrounding mesenchymal cells and the extracellular matrix (Davis etal., 1996; Suri et al., 1996). Arai and collaborators (2004),demonstrated that Ang1 enhances through Tie2 the adhesion of HSCs to beadhered to the bone. Since Tie2 is expressed in human glioma cells, theinventors contemplate that Tie2 is critical for maintenance of theneoplastic phenotype via cell adhesion to the ECM of the tumor.Therefore, the effects of Tie2/Ang-1 signaling on cell adhesion willanalyzed.

First Tie2+ cell lines (U-87 MG and A172) will be plated onto wells of atissue culture dish that had been precoated with either Ang-1 or Ang-2or various other known ECM proteins. These experiments will be performedalso with isogenic cell lines that differ only in their Tie2 status(U-251 MG). The requirement of Tie2 for adhesion will be assessed byblocking the positive reactions with sTie2 and or T4 peptide. Because ithas been recently demonstrated that Tie2/Ang-1 interaction enhancesadhesion of HSC via β1-integrin (Arai et al., 2004), the effect of theAng1-treatment of Tie-2+ human glioma cells U-87 MG, A172, andTie2-expressing U-251 MG in the expression of β1-integrin will bestudied using Western blot. If β1-integrin is upregulated upon Tie2activation, adhesion studies will be performed using RGD peptide asblocker. In a parallel set of studie, pharmacological inhibition ofERK1/2 activation (PD98059) will be induced and assessed forAng1-induced adhesion, suggesting that intracellular activation of MAPKis required for Ang1-induced adhesion.

Another study is based on the reported capability of Ang1 to beincorporated into the ECM (Xu et al. 2001). To understand the impact ofthat phenomena in glioma phenotype, ECM-associated Ang1 will be studiedin glioma adhesion. Glioma cells will be seeded onto plastic dishes withor without Ang1 or the culture dish containing the ECM componentsdeposited by confluent U-87MG expressing Ang1 (infected with AdAng1).Adherence of glioma cells to ECM-associated Ang1, as well as theactivation of Tie2 receptor in their surfaces will be assessed bywestern blot. Competitive experiments will include the blockade of theTie2 receptor binding (sTie2, T4), as well as integrin binding (RGD).

The role of Tie2/Ang1 in the migration of glioma cells. Several reportshave shown data on the chemotactic properties of Ang1 for endothelialcells (Witzenbichler et al, 1998), as well as eosinophils (Feistritzeret al., 2004). An isogenic system (U-87parental and siTie2-U87 MG; U-251MG parental and Tie2-expressing U-251 MG) will be used to testchemotactic properties of Ang1 in cancer cells, particularly gliomas.Using chemotaxis Boyden chambers, the chemotactic response of those celllines to increasing amounts of Ang1 and Ang-2 will be assessed andcompared to that FBS. To establish specificity, Ang1 will be applied atdifferent doses with and without a 10-fold excess to reach maximalsaturated concentration of either sTie2 or T4 to the lower chambers. Todistinguish between chemotactic and chemokinetics effects of Ang1 onglioma cells, a modified checkerboard analysis will be performed inwhich the concentration of the chemoattractant above and below thefilter will vary. If Ang1 is a chemoattractant factor, glioma cells onlywill migrate when a concentration gradient is present.

Generation of an oncolytic adenovirus retargeted to infect Tie2+ cells.For uptake-related studies, the inventors have constructed isogenic celllines that only differ in their Tie2 status. The inventors alsocontemplate the introduction of GFP cDNA in a retargeted Tie2 constructto easily monitor the adenoviral infectivity. In order to examine theselective replication of the proposed construct, the MAPK activity ofseveral cell lines (Ahmed et al., 2003) have been tested and will useMAPK inhibitors for specificity questions.

Generation of Ad-E1A-COX-Tie2. To study uptake of a retargetedadenovirus, the inventors will construct an adenovirus carrying the GFPcDNA in the E3 deleted region, driven by the CMV promoter. T4 (Tie2ligand) coding sequence will be inserted in the HI loop of the fiberprotein. The modified adenoviruses will be propagated in A549 cells(ATCC) where the virions should have only chimeric fibers. Since A549cells do not express adenoviral genes, the production of wild-typeadenovirus is highly unlikely. PCR amplification of E1A, and knobfiber-encoding sequence, followed by enzyme digestion (Fueyo et al.,2000) will be performed for each batch of the virus (See Fueyo et al.,2003). An Ad-E1A-cTie2 will be generated as a control, having a similarbackbone structure than Ad-E1A-Tie2 but having a random peptide in theHI loop. Both adenoviruses will carry a minicassette of expression forthe GFP gene (CMV-GFP-SV40 polyadenylation signal) that will be clonedin the deleted E3 region. The strategy will consist of introducing theTie2 ligand into the HI loop of the fiber protein. pXKdeltaHI is a fibershuttle vector designed to introduce the ligands into the HI loop of thefiber protein and will be used in the construction of retargetedadenoviruses of the invention. The T4 peptide encoding sequence will becloned into the EcoRV site of the vector. In addition to theligand-encoding sequence DNA fragment to be cloned should contain twoThr codons upstream from the ligand and a CC sequence downstream of it(Thr-Thr-ligand-CC). This way, the ligand will be positioned between theThr-546 and Pro-547 residues of the fiber protein, that is—within the HIloop sequence. For CAR-binding ablation the approach by Alemany et al.(2001) will followed, mutating the residue Y477 in the loop of thefiber, which is critical for binding.

Replication-selective Tie2 targeted adenovirus. In a second step theinventor will construct the Ad-E1A-COX-Tie2. Ad-E1A-COX-Tie2 willcontain the adenovirus E1A gene fused with the PTGS2 3′ UTR, as Ahmed etal., 2003. For this construct the inventors will repeat several of thein vitro experiments performed with Ad-E1A-Tie2, and then it will betested for anti-cancer effect in vivo. Adenoviral constructs used ascontrols will include adenovirus with a mutant peptide (cTie2) in the HIloop (Ad-E1A-Cox-cTie2), Ad-E1A-Cox (non-retargeted virus), Adwt,Adwt-Tie2, and PBS. For the in vivo experiments isogenic systems fromhigh-MAPK activity cell lines (U-87 MG and U-251 MG) will be used, aswell as low-MAPK activity cell line (U-118 MG). The patologicexamination of the infected xenografts should reveal the characteristicsof the adenoviral spread, the timing of the spread and the pathologicbasis of the anti-glioma effect.

Selective Infectivity of the CAR-Ablated Tie2-targeted Adenoviruses. Inorder to ascertain whether the insertion of the Tie2 fiber motif isresponsible for selective infectivity, two studies will be performedwith an established system where U-251 MG cells have been stablytransfected with Tie2 cDNA. In addition, infectivity experiments will beperformed in a set of cell lines that express different levels of Tie2and CAR: HUVEC, U-87 MG, A172 (low CAR/high Tie2), LN229, SNB19 (highCAR, low Tie2).

CAR-ablated Ad-E1A-Tie2 infection via Tie2. The inventors will use GFP(cell quantification) and E1A expression (cell quantification byimmunohistochemistry) to quantified the infectivity of the differentconstructs. The inventors contemplate that the Tie2-targeted vector willinfect high-Tie2 expressing cell lines more efficiently, independentlyof their CAR expression. The Ad-cTie2 construct, which is expressing inthe HI loop of the fiber the control peptide, should infect all thesecultures poorly.

Mechanisms of Ad-Tie2 uptake. Competitive inhibition of Ad-mediated genedelivery by the T4 peptide (Tie2) will be studied using test and controladenoviruses in assays based on the competitive inhibition ofAd-mediated gene delivery by the targeted Tie2 peptide (T4)(see Fueyo etal., 2003 for methods). These studies will address the specificity ofthe Tie2-adenovirus construct. Consequently, cells will be treated witheither T4 or a control peptide, cT4, and then infected with Ad-GFP-Tie2,Ad-GFP-cTie2, or wild-type adenoviruses. Since Tie2 is used only by theAd-Tie2 construct for internalization, the inventors contemplate adecrease in the infectivity of this vector in the U-251-Tie2 cellspretreated with the T4 peptide. On the contrary, pretreatment with thispeptide should not result in a modification of the infectivity of thecontrol adenoviruses.

Viral production. Ad-GFP-Tie2 and Ad-GFP-cTie2 possess similarreplication systems but the insertion of the Tie2-targeted peptide andablation of the CAR binding enhances the selectivity of the former toinfect Tie2 expressing glioma cells. Since the ability to replicate isinherently related to the ability of the virus to infect, the inventorsare interested in determining whether Ad-GFP-Tie2 infection leads to anincreased viral production in Tie2-expressing cancer cells. For viralproduction analysis the inventors will use TCID50 methodology and anhexon-expressing quantitative assay (BD Bioscience).

Oncolytic power of Ad-E1A-Cox-Tie2. Studies will be performed to comparethe oncolytic effects of Ad-E1A-Cox-Tie2, Ad-E1A-Cox-cTie2, Ad-E1A-Cox(non-retargeted virus), Adwt, Adwt-Tie2 in normal (toxicity) and cancercell cultures. These studies will be design to demonstrate that the newconstruct enhances oncolytic effect in the cultures with Tie2 and lowCAR expression (U-87 MG and HUVEC).

Cell viability analyses. Crystal Violet and Trypan Blue Exclusion Testwill be performed in the panel of glioma cells described above afterinfection with Ad-E1A-Cox-Tie2, and the explained controls. Dosedependence experiments will be performed (Gomez-Manzano et al., 2004).

Toxicity in normal cultures. The expression of Tie2 and CAR in NormalHuman astrocytes and Neuronal Precursors will be assessed by RT-PCR,Western blot and Immunohistochemical analyses. Transduction will beassessed by GFP detection analyzed after infection with theGFP-constructs (Ad-GFP-Tie2, Ad-GFP-cTie2, and Ad-GFP). Cell viabilitywill be assessed by trypan blue exclusion test after viral infection.The inventors contemplate that although neuronal precursors expressTie2, these cells would not support a efficient viral replication due tolow levels of MAPK activity.

MAPK-dependent replication. Although the correlation between MAPKactivity and Ad-E1A-COX ability to replicate has been shown (Ahmed etal., 2003), the inventors have designed studies to assess the ability ofretargeted constructs to replicate in glioma cells with high or low MAPKactivity. To characterize the effects of the insertion of the 3′ UTR onE1A expression, inhibitors of kinases signaling, PD98059 (p42/p44 MAPKinhibitor), and for specificity testing: SB203580 (p38 MAPK inhibitor)and LY294002 (PI3K inhibitor) will be used. The inventors will use anisogenic U-87 MG system, containing a stably integrated, IPTG-inducibleactivated Ha-rasVal12 cDNA (Sheng et al., 2000). This model system isbased in cells that only differ in the expression of an activatedvariant of the Hras oncogene, Ha-rasVal12 (Sheng et al., 2000). The willassess whether PTGS2 3′ UTR-mediated Ad-E1A-COX-Tie2 depends on theP-MAPK pathway. The inventors have shown that PD98059 effectivelyinhibited P-MAPK expression in U-87 MG cells. The replication capabilityof Ad-E1A-COX-Tie2 in U87-MG cells untreated and treated with PD98059(and the control inhibitors) will be assessed. The inventors contemplatethat inhibition of P-MAPK activity by PD98059 will greatly reduce theability Ad-E1A-COX-Tie2 to replicate even with high expression ofHa-rasVal12 induced by IPTG in the isogenic U-87 MG. To further assessthe selectivity of the Ad-E1A-COX-Tie2 virus, the inventors will usehistologically closely matched glioma tumor lines that differ in P-MAPKactivity (Ahmed et al., 2003).

Correlation of Tie2 targeted therapy and survival. The implantableguide-screw method, as described herein will be used in these studies(Lal et al., 2000). In vivo experiments will be performed with 3isogenic cell line-based systems expressing different levels of Tie2:(a) parental U-87 MG (high expression levels of Tie2 and high MAPKactivity) and siTie2-transfected U-87 MG cells (low Tie2 expression);(b) U-251 MG parental (undetectable levels of Tie2 and high MAPKactivity) and U-251 MG-Tie2 (high expression levels of Tie2 and highMAPK activity); and (c) U-118 MG parental (low MAPK, Tie2) and U-118isogenic cell line (low MAPK, Tie2). The studies with the U-87 MG system(very low CAR expression) address the question of the relevance of Tie2on the anti-cancer effect. U-87 MG parental cells will be implanted intothe brain of nude mice animals (Lal et al., 2000), and then treated withAd-E1A-Cox-Tie2 (T4 peptide), Ad-E1A-Cox-cTie2 (control peptide),Ad-E1A-Cox (non-retargeted virus), Adwt, Adwt-Tie2, and PBS (to examinethe effect of the technique in survival). A similar strategy will beused with the isogenic cell line U-87 MG.siTie2 cells. Results frompreliminary studies show that an empirically chosen dose of 1.5×10⁸(administered three times) of a non-targeted and retargeted constructsignificantly improves survival time (Fueyo et al., 2003; Gomez-Manzanoet al., 2004). Animals will be sacrificed if and when they demonstratesigns of general toxicity, or neurological signs or, in the case of longsurvivors, animals will be euthanized 90 days post-implantation, andbrains will be extracted. Tumors will be examined using H/E staining andimmunohistochemistry for Tie2 and p42/p44 MAPK (basal and phosphorylatedlevels) expression. Similar experiments will be perforrned with thethree above described pair of isogenic cell lines.

One of skill in the art readily appreciates that the present inventionis well adapted to carry out the objectives and obtain the ends andadvantages mentioned as well as those inherent therein. Methods,procedures and techniques described herein are presently representativeof the preferred embodiments and are intended to be exemplary and arenot intended as limitations of the scope. Changes therein and other useswill occur to those skilled in the art which are encompassed within thespirit of the invention or defined by the scope of the pending claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 3,817,837-   U.S. Pat. No. 3,850,752-   U.S. Pat. No. 3,939,350-   U.S. Pat. No. 3,996,345-   U.S. Pat. No. 4,196,265-   U.S. Pat. No. 4,275,149-   U.S. Pat. No. 4,277,437-   U.S. Pat. No. 4,366,241-   U.S. Pat. No. 4,472,509-   U.S. Pat. No. 4,554,101-   U.S. Pat. No. 4,683,195-   U.S. Pat. No. 4,683,202-   U.S. Pat. No. 4,800,159-   U.S. Pat. No. 4,883,750-   U.S. Pat. No. 4,938,948-   U.S. Pat. No. 4,946,773-   U.S. Pat. No. 5,021,236-   U.S. Pat. No. 5,196,066-   U.S. Pat. No. 5,279,721-   U.S. Pat. No. 5,545,548-   U.S. Pat. No. 5,665,567-   U.S. Pat. No. 5,840,873-   U.S. Pat. No. 5,843,640-   U.S. Pat. No. 5,843,650-   U.S. Pat. No. 5,843,651-   U.S. Pat. No. 5,846,708-   U.S. Pat. No. 5,846,709-   U.S. Pat. No. 5,846,717-   U.S. Pat. No. 5,846,726-   U.S. Pat. No. 5,846,729-   U.S. Pat. No. 5,846,783-   U.S. Pat. No. 5,849,483-   U.S. Pat. No. 5,849,487-   U.S. Pat. No. 5,849,497-   U.S. Pat. No. 5,849,546-   U.S. Pat. No. 5,849,547-   U.S. Pat. No. 5,851,770-   U.S. Pat. No. 5,853,990-   U.S. Pat. No. 5,853,992-   U.S. Pat. No. 5,853,993-   U.S. Pat. No. 5,856,092-   U.S. Pat. No. 5,858,652-   U.S. Pat. No. 5,861,244-   U.S. Pat. No. 5,863,732-   U.S. Pat. No. 5,863,753-   U.S. Pat. No. 5,866,331-   U.S. Pat. No. 5,866,337-   U.S. Pat. No. 5,866,366-   U.S. Pat. No. 5,882,864-   U.S. Pat. No. 5,905,024-   U.S. Pat. No. 5,910,407-   U.S. Pat. No. 5,912,124-   U.S. Pat. No. 5,912,145-   U.S. Pat. No. 5,912,148-   U.S. Pat. No. 5,916,776-   U.S. Pat. No. 5,916,779-   U.S. Pat. No. 5,919,630-   U.S. Pat. No. 5,922,574-   U.S. Pat. No. 5,925,517-   U.S. Pat. No. 5,925,525-   U.S. Pat. No. 5,928,862-   U.S. Pat. No. 5,928,869-   U.S. Pat. No. 5,928,870-   U.S. Pat. No. 5,928,905-   U.S. Pat. No. 5,928,906-   U.S. Pat. No. 5,929,227-   U.S. Pat. No. 5,932,413-   U.S. Pat. No. 5,932,451-   U.S. Pat. No. 5,935,791-   U.S. Pat. No. 5,935,825-   U.S. Pat. No. 5,939,291-   U.S. Pat. No. 5,942,391-   U.S. Pat. No. 6,586,411-   U.S. patent application 20030138405-   Abbondanzo et al., Breast Cancer Res. Treat., 16:182(#151), 1990.-   Ahmed et al., Nat. Biotechnol., 21(7):771-777, 2003.-   Aiello, Proc. Natl. Acad. Sci. USA, 92:10457, 1995.-   Alemany et al., Cancer Gene Ther., 6:21-5, 1999.-   Alemany et al., Neurologia., 16(3): 122-127, 2001.-   Allred et al., Arch. Surg., 125(1):107-113, 1990.-   Andreanski et al., Cancer Res., 57:1502-1509, 1997.-   Arai etal., Cell, 118(2):149-161, 2004.-   Atherton et al., Biol. Reprod., 32(1):155-171, 1985.-   Audero etal., J. Biol. Chem., 279(13):13224-13233, 2004.-   Bellus, J. Macromol. Sci. Pure Appl. Chem., A31(1): 1355-1376, 1994.-   Bernt et al., J. Virol., 76:10994-1002, 2002.-   Bigner et al. Cancer Res., 50(24):8017-8022, 1990.-   Bilton and Booker, Eur. J. Biochem., 270:791-798, 2003.-   Bischoff et al., Science, 274:373-376, 1996.-   Brooks et al., Methods Mol Biol., 129:257-69, 1999.-   Brown et al. Immunol. Ser., 53:69-82, 1990.-   Brown et al., Am. J. Pathol., 156: 2179-2183, 2000.-   Cameliet and Jain, Nature, 407:249-57, 2000.-   Campa et al., Biochem. Biophys. Res. Commun., 275(2):631-636., 2000.-   Carlson et al., J. Biol. Chem., 276(28):26516-26525, 2001.-   Carlson et al., Mol. Ther., 6:99-105, 2002.-   Chase et al., Nat. Biotechnol., 16, 444-448, 1998.-   Cheng et al., Proc. Natl. Acad. Sci. USA, 93:8502-8507, 1996.-   Chintala et al., Oncogene, 15( 17):2049-2057, 1997.-   Cho et al., Gene Ther., 7(9):740-749, 2000.-   Cho et al., Gene Ther., 9(17):1139-1145, 2002.-   Coffeyetal., Science, 282:1332-1334, 1998.-   Collins and James, FASEB J., 7:926-930, 1993.-   Costello et al., Cancer Res., 56:2405-2410, 1996.-   Costello et al., Cancer Res., 57:1250-1254, 1997.-   Dai et al., Nature, 379:458-460, 1996.-   Davis etal., Cell, 87:1161-116, 1996.-   Davis et al., Clin. Cancer Res., 10:33-42, 2004.-   De Jager et al., Semin. Nucl. Med., 23(2):165-179, 1993.-   DeBusk et al., Exp. Cell. Res., 298(1):167-177, 2004.-   Dholakia et al., J. Biol. Chem., 264(34):20638-20642, 1989.-   Doolittle and Ben-Zeev, Methods Mol Biol, 109:215-237, 1999.-   Drapkin et al., J. Clin. Invest., 105(5):589-596, 2000.-   Dyson and Harlow, Cancer Surv., 12:161-195, 1992.-   Ekstrand et al., Proc. Natl. Acad. Sci. USA, 89(10):4309-4313, 1992.-   Eskandar et al., Anat, Rec., 230(2):169-174, 1991.-   European Appln. 320308-   European Appln. 329 822-   Feistritzer et al., J Allergy Clin Immunol., 114(5):1077-1084, 2004.-   Ferrara and Alitalo, Nature Medicine, 5:1359-1364, 1999.-   Ferrara, Nat. Rev. Cancer, 2:795-803, 2002.-   Fine et al., J. Clin. Oncol., 18:708-715, 2000.-   Flint and Shenk, Annu. Rev. Genet., 31:177-212, 1997.-   Forsythe et al., Mol. Cell Biol., 16:4604-4613, 1996.-   Frederick et al., Neuro-oncol., 2(3): 159-163, 2000.-   Freytag et al., Hum. Gene Ther., 9:1323-1333, 1998.-   Frohman, In: PCR Protocols: A Guide To Methods And Applications,    Academic Press, N.Y., 1990.-   Fueyo et al., Archives of Neurology, 56(4):445-448, 1999.-   Fueyo et al., J. Natl. Cancer Inst., 95:652-60, 2003.-   Fueyo et al., Nat. Med., 4:685-690, 1998b.-   Fueyo et al., Nature Medicine, 4(6):685-690, 1998.-   Fueyo et al., Neurology, 50:1307-1315, 1998c.-   Fueyo et al., Neurology, 51:1250-1255, 1998a.-   Fueyo et al., Oncogene, 12:103-110, 1996a.-   Fueyo et al., Oncogene, 13:1615-1619, 1996b.-   Fueyo et al., Oncogene, 19:2-12, 2000.-   Fueyo et al., Oncogene, 19:2-12, 2000.-   Fujikawa et al., J. Vasc. Res., 36(4):272-281, 1999.-   Fumari et al., Cancer Surv., 25, 233-275, 1995.-   Gage, Obstet. Gynecol., 96(6):879-885, 2000.-   Galli et al., 2004-   GB Appln. 2 202 328-   Geoerger et al., Cancer Res., 62(3):764-772, 2002.-   Ghosh and Bachhawat, In: Liver Diseases, Targeted Diagnosis and    Therapy Using Specific Receptors and Ligands, Wu et al. (Eds.),    Marcel Dekker, N.Y., 87-104, 1991.-   Gomez-Manzano et al., Ann. Neurol., 53:109-17, 2003.-   Gomez-Manzano et al., Cancer Res., 56:694-9, 1996.-   Gomez-Manzano et al., In: Gene Transfer and Therapy for Neurological    Disorders, Chiocca and Breakefield (Eds.), Human Press Inc.: N.J.,    201-225, 1998.-   Gomez-Manzano et al., Int. J. Oncol., 19(2):359-365, 2001.-   Gomez-Manzano et al., Oncogene, 23(10):1821-1828, 2004.-   Graham and Prevec, In: Methods in Molecular Biology: Gene Transfer    and Expression Protocol, Murray (Ed.), Humana Press, Clifton, N.J.,    7:109-128, 1991.-   Grunhaus and Horwitz, Seminar in Virology, 3:237-252, 1992.-   Gulbis and Galand, Hum. Pathol., 24(12):1271-1285, 1993.-   Haberkorn and Altmann, Curr. Gene Ther., 1(2):163-182, 2001.-   Haberkorn and Altmann, J. Cell Biochem. Suppl., 39:1-10, 2002.-   Haberkorn et al., Eur. J. Nucl. Med., 28(5):633-638, 2001.-   Haberkorn et al., J. Nuc. Med., 42(2):317-325, 2001.-   Haberkorn, Exp. Clin. Endocrinol. Diabetes, 109(1):60-62, 2001.-   Hamel et al., J. Neurooncol., 16:159-165, 1993.-   Hamstra et al., Hum. Gene Ther., 10:1993-2003, 1999.-   Harlow and Lane, In: Antibodies: A Laboratory Manual, Cold Spring    Harbor Laboratory, Cold Spring Harbor, N.Y., 346-348, 1988.-   Heise et al., Nat. Med., 3:639-645, 1997.-   Heise et al., Nat. Med., 6:1134-9, 2000.-   Hemminki et al. Cancer Res., 61(17):6377-6381, 2001.-   Henson et al., Ann. Neurol., 36:714-721, 1994.-   Hermiston, J. Clin. Invest., 105:1169-1172, 2000.-   Hess et al., Neuro-Oncol., 1(4):282-288, 1999.-   Hirvonen et al., Br. J. Cancer, 69:16-25, 1994.-   Holashetal., Oncogene, 18:5356-62, 1999b.-   Holash et al., Science, 284:1994-8, 1999a.-   Im et al., Br. J. Cancer, 84:1252-1252, 2001.-   Im et al., Cancer Res., 1999, 59:895-900, 1999.-   Innis et al., PCR Protocols, Academic Press, Inc., San Diego Calif.,    1990.-   Ipata et al., Biochemistry, 10(23):4270-4276, 1971.-   Ipata et al., Methods Enzymol., 51:394-400, 1978.-   Jacotot, Acad. Sci. Hebd. Seances Acad. Sci., 264(22):2602-2603,    1967.-   Jen et al., Cancer Res., 54:6353-6358, 1994.-   Jin et al., Cancer Res., 60(5):1221-1224, 2000.-   Johnson et al., 1994-   Jones and Dumont, Oncogene, 17(9):1097-1108, 1998.-   Jones et al., Mol. Cell Biol., 23(8):2658-2668, 2003.-   Kaelin, Bioessays, 21(11):950-8, 1999.-   Karlsson et al., EMBO J., 5:2377-2385, 1986.-   Katsuragi et al., Appl. Biochem. Biotechnol., 16:61-69, 1987.-   Katsuragi et al., Exp. Pathol., 29(3):129-142, 1986.-   Ke et al., Cancer Res.,. 62:1854-1861, 2002.-   Ke et al., Clin. Cancer Res., 6: 2562-2562, 2000b.-   Ke et al., Mol. Cell Probes, 14:127-135, 2000a.-   Khatoon et al., Ann. Neurol., 26(2):210-5, 1989.-   Kievitetal., Cancer Res., 59:1417-1421, 1999.-   Kim et al., FASEB J., 16:1126-1128, 2002.-   Kim et al., Proc. Natl. Acad. Sci. USA, 99:11399-11404, 2002.-   King et al., J. Biol. Chem., 264(17):10210-10218, 1989.-   Kim et al., Nat. Med., 4, 1341-1342, 1998.-   Kim, Expert Opin. Biol. Ther., 1(3):525-538, 2001.-   Koblizek et al., Curr Biol., 8(9):529-532, 1998.-   Kohrle, Z rztl Fortbild Qualitatssich, 93(1):17-22, 1999.-   Kontos et al., Mol. Cell Biol., 18(7):4131-4140, 1998.-   Krasnykh et al., J. Virol., 75(9):4176-83, 2001.-   Kruse et al., J. Neuro-Oncol., 19:161-168, 1994.-   Kuan et al., Endocr. Relat. Cancer, 8(2):83-96, 2001.-   Kuan et al., Endocr. Relat. Cancer, 8(2):83-96, 2001.-   Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173, 1989.-   Kyritsis and Yung, Baillieres Clinical Neurology, 5(2):295-305,    1996.-   Kyritsis et al., Mol. Carcinog., 15:1-4, 1996b.-   Kyritsis et al., Oncogene, 12:63-67, 1996a.-   Kyte and Doolittle, J. Mol. Biol., 157(1):105-132, 1982.-   La Perle et al., Prostate, 50(3):170-178, 2002.-   Lal et al., J. Neurosurg., 92:326-333, 2000.-   Lal et al., J. Neurosurgery, 92:326-333, 2000.-   Lang et al., J. Clin. Oncol.,;21:2508-2518, 2003.-   Levine, JAMA, 273(7):592, 1995.-   Levy et al., J. Bioenerg. Biomembr., 30(2):195-206, 1998b.-   Levy et al., J. Biol. Chem., 273(35):22657-22663, 1998a.-   Lin et al., Proc. Natl. Acad. Sci. USA, 95:8829-34, 1998.-   Lorimer et al., Proc. Natl. Acad. Sci. USA, 93(25):14815-14820,    1996.-   Mabjeesh et al., Cancer Cell, 3:363-75, 2003.-   Macejak and Samow, Nature, 353:90-94, 1991.-   Maisonpierre et al., Science, 277:55-60, 1997.-   Manley et al., Eur. J. Cancer., 38(5):S19-27, 2002.-   Martuza et al., Science, 10:854-856, 1991.-   Master et al., 2001-   McCarthy et al., J. Cell Biol., 85(3):890-902, 1980.-   Millauer et al., Nature, 367:576-9, 1994.-   Miller et al., Cancer Res. 62:773-780, 2002.-   Mineta et al., Nat. Med., 9:938-943, 1995.-   Mishima et al., Cancer Res., 61(14):5349-5354, 2001.-   Nakamura et al., In: Handbook of Experimental Immunology (4^(th)    Ed.), Weir et al. (Eds), 1:27, Blackwell Scientific Publ., Oxford,    1987.-   Nevins, Science, 258:424-429, 1992.-   Nishikawa et al. Proc. Natl. Acad. Sci. USA, 91(16):7727-7731, 1994.-   Nishiyama et al., Cancer Res., 45(4):1753-1761, 1985.-   Niu et al., J. Biol. Chem., 277(35):31768-31773, 2004.-   O'Shannessy et al., Anal Biochem, 163(1):204-209, 1987.-   O'Donovan and Neuhard, Bacteriol. Rev., 34(3):278-343, 1970.-   Ohara et al., Proc. Natl. Acad. Sci. USA, 86:5673-5677, 1989.-   Owens and Haley, Biochem. Biophys. Re.s Commun., 142(3):964-971,    1987.-   Papapetropoulos et al., J. Biol. Chem., 275(13):9102-91025 , 2000.-   PCT Appln. PCT/US87/00880-   PCT Appln. PCT/US89/01025-   PCT Appln. WO 88/10315-   PCT Appln. WO 89/06700-   PCT Appln. WO 95/27071-   PCT Appln. WO 96/12406-   PCT Appln. WO 96/33280-   PCT Appln. WO 90/07641-   Pelletier and Sonenberg, Nature, 334(6180):320-325, 1988.-   Petrich et al., Eur. J. Nucl. Med. Mol. Imaging, 29(7):842-854,    2002.-   Plate et al. 1992-   Pollack et al., J. Neurosurg., 73(1):106-112, 1990.-   Poncet et al., Neuropathol. Appl. Neurobiol., 29:361-9, 2003.-   Potter and Haley, Methods Enzymol, 91:613-633, 1983.-   Puumalainen et al., Hum. Gene Ther., 9:1769-1774, 1998.-   Racher et al., Biotechnology Techniques, 9:169-174, 1995.-   Remington's Pharmaceutical Sciences, 15^(th) ed., 33:624-652, Mack    Publishing Company, Easton, Pa., 1980.-   Reynolds et al., 1996-   Roth and Cristiano. J. Natl. Cancer Inst., 89:21-39, 1997.-   Rouslahti and Rajotte, Annu. Rev. Immunol., 18, 813-827, 2000.-   Sambrook et al., In: Molecular cloning, Cold Spring Harbor    Laboratory Press, Cold Spring Harbor, N.Y., 2001.-   Sato et al., Int. Immunol., 10(8):1217-1227, 1998.-   Sato et al., Nature, 376:70-74, 1995.-   Schiffer, Forum, 8(3):244-255, 1998.-   Schmidt et al., Cancer Res., 54:6321-6324, 1994.-   Sehgal, Seminars in Surgical Oncology, 14(1): 3-12, 1998.-   Semenza, Biochem. Pharmacol., 64:993-8, 2002.-   Senter et al., Bioconjug. Chem., 2(6):447-451, 1991.-   Shapiro and Shapiro, Oncology, 12(2):233-240, 1998.-   Sheng et al., Oncogene, 19(42):4847-4854, 2000.-   Sheta et al., Oncogene, 20:7624-34, 2001.-   Sidransky et al., Nature, 355:846-847, 1992.-   Singh 2004-   Singh et al., 2003-   Smanik et al., Biochem. Biophys. Res. Commun., 226:339-345, 1996.-   Springer et al., Mol Ther., 1(1):82-87, 2000.-   Steinwaerder et al., Nat. Med., 7:240-3, 2001.-   Sugawa et al. Proc. Natl. Acad. Sci. USA, 87(21):8602-5606, 1990.-   Suri et al., Cell, 87:1171-80, 1996.-   Suri et al., Science, 282:468-71, 1998.-   Suzuki et al., Clin. Cancer Res., 7:120-126, 2001.-   Suzuki et al., Clin. Cancer Res., 7:120-6, 2001.-   Takakura et al., Immunity, 9(5):677-686, 1998.-   Thomas et al., Semin. Oncol., 30:32-8, 2003.-   Toumaire et al., EMBO Rep., 5(3):262-267, 2004.-   Uekietal., Cancer Res., 56:150-153, 1996.-   Valable et al., FASEB J., 17:443-5, 2003.-   Valk-Lingbeek et al., Cell, 11 8(4):409-418 , 2004.-   Vile, Cancer Gene Ther., 9(12):1062-1067, 2002.-   Wang et al., 2004-   Wang et al., Biotechniques, 31:196-202, 2001.-   Weietal., Clin. Cancer Res., 1(10):1171-1177, 1995.-   West et al., J. Bacteriol., 149(3):1171-1174, 1982.-   Whyte et al., Cell, 56:67-75, 1989.-   Whyte et al., Cell, 62:257-265, 1988.-   Wildner et al., Cancer Res., 59:410-413, 1999.-   Witzenbichler et al., J. Biol. Chem., 273(29):18514-18521, 1998.-   Xu et al., 2001-   Yamaguchi et al., 1994-   Yan et al., J. Virol., 77(4):2640-2650, 2003.-   Yancopoulos et al., Nature, 407: 242-8, 2000.-   Yergatian et al., Experientia, 33(12):1570-1571, 1977.-   Yoon et al., Biochem. Biophys. Res. Commun., 308:101-5, 2003.-   Zadeh et al., Am. J. Pathol., 164:467-76, 2004.-   Zagzag et al., Lab. Invest., 80:837-49, 2000.-   Zoltan et al., 1996

1. A method for treating a brain tumor in a patient comprising: a)identifying a patient having a brain tumor; and b) contacting the tumorwith an oncolytic adenovirus that encodes a therapeutic gene and an E1Apolypeptide that cannot bind Rb.
 2. The method of claim 1, wherein theoncolytic adenovirus is a Delta 24 adenovirus.
 3. The method of claim 1,wherein the oncolytic adenovirus comprises a targeting moiety.
 4. Themethod of claim 3, wherein the targeting moiety is a chimeric adenoviralfiber protein.
 5. The method of claim 4, wherein the chimeric adenoviralfiber protein comprises an RGD amino acid sequence.
 6. The method ofclaim 4, wherein the chimeric adenoviral fiber protein comprises a vIIIamino acid sequence.
 7. The method of claim 4, wherein the chimericadenoviral fiber protein comprises a PEPHC1 amino acid sequence.
 8. Themethod of claim 1, wherein the therapeutic gene is Rb, CFTR, p16, p21,p27, p57, p73, C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1,MEN-I, MEN-II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF, G-CSF,thymidine kinase, mda7, fus, interferon α, interferon β, interferon γ,ADP, p53, ABL1, BLC1, BLC6, CBFA1, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1,ETS2, ETV6, FGR, FOX, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL,MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3, YES,MADH4, RB1, TP53, WT1, TNF, BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3,NT5, ApoAI, ApoAIV, ApoE, Rap1A, cytosine deaminase, Fab, ScFv, BRCA2,zac1, ATM, HIC-1, DPC-4, FHIT, PTEN, ING1, NOEY1, NOEY2, OVCA1, MADR2,53BP2, IRF-1, Rb, zac1, DBCCR-1, rks-3, COX-1, TFPI, PGS, Dp, E2F, ras,myc, neu, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, VEGF, FGF,thrombospondin, BAI-1, GDAIF, or MCC.
 9. The method of claim 1, whereinthe therapeutic gene is yeast cytosine deaminase.
 10. The method ofclaim 1, wherein the therapeutic gene is Ang-2.
 11. The method of claim1, wherein the therapeutic gene is NIS.
 12. The method of claim 1,wherein the tumor is contacted with the adenovirus by delivery of theadenovirus intracranially into the patient.
 13. The method of claim 1,wherein the tumor is a astrocytoma, oligodendroglioma, anaplasticglioma, glioblastoma, ependymoma, meningioma, pineal region tumor,choroid plexus tumor, neuroepithelial tumor, embryonal tumor, peripheralneuroblastic tumor, tumor of cranial nerves, tumor of the hemopoieticsystem, germ cell tumors, or tumor of the sellar region.
 14. The methodof claim 13, wherein the tumor is a glioblastoma.
 15. A method oftreating cancer in a patient comprising: a) administering to a patientan effective amount of a composition comprising an oncolytic adenoviruscomprising (i) a nucleic acid, wherein the nucleotides encoding aminoacids 122-129 of the encoded E1A polypeptide are deleted, and (ii) anexpression cassette comprising a polynucleotide encoding a yeastcytosine deaminase; and b) administering an effective amount of apro-drug, wherein the pro-drug is metabolized to a cytotoxic drug by apolypeptide encoded by the yeast cytosine deaminase.
 16. The method ofclaim 15, wherein the yeast cytosine deaminase is a humanized yeastcytosine deaminase.
 17. The method of claim 15, wherein the oncolyticadenovirus comprises a targeting moiety.
 18. The method of claim 17,wherein the targeting moiety is a chimeric adenoviral fiber protein. 19.The method of claim 17, wherein the chimeric adenoviral fiber proteincomprises an RGD amino acid sequence.
 20. The method of claim 18,wherein the chimeric adenoviral fiber protein comprises a vIII aminoacid sequence.
 21. The method of claim 18, wherein the chimericadenoviral fiber protein comprises a PEPHC1 amino acid sequence.
 22. Themethod of claim 16, wherein the cell is a tumor cell.
 23. The method ofclaim 16, wherein the cancer is a astrocytoma, oligodendroglioma,anaplastic glioma, glioblastoma, ependymoma, meningioma, pineal regiontumor, choroid plexus tumor, neuroepithelial tumor, embryonal tumor,peripheral neuroblastic tumor, tumor of cranial nerves, tumor of thehemopoietic system, germ cell tumors, or tumor of the sellar region. 24.The method of claim 23, wherein the tumor is a glioblastoma.
 25. Themethod of claim 15, wherein the oncolytic adenovirus is suitablydispersed in a pharmacologically acceptable formulation.
 26. The methodof claim 15, wherein the composition is administered intracranially. 27.The method of claim 26, wherein the composition is directly injectedinto a tumor.
 28. The method of claim 26, wherein the administrationoccurs more than once.
 29. The method of claim 28, wherein thecomposition is administered at least three times to the patient.
 30. Themethod of claim 15, further comprising administering to the patient asecond therapy, wherein the second therapy is anti-angiogenic therapy,chemotherapy, immunotherapy, surgery, radiotherapy, immunosuppresiveagents, or gene therapy with a therapeutic polynucleotide.
 31. Themethod of claim 30, wherein the second therapy is administered to thepatient before administration of the composition comprising theoncolytic adenovirus.
 32. The method of claim 30, wherein the secondtherapy is administered to the patient at the same time asadministration of the composition comprising the oncolytic adenovirus.33. The method of claim 30, wherein the second therapy is administeredto the patient after administration of the composition comprising theoncolytic adenovirus.
 34. The method of claim 30, wherein thechemotherapy comprises an alkylating agent, mitotic inhibitor,antibiotic, or antimetabolite.
 35. The method of claim 30, wherein thechemotherapy comprises CPT-11, temozolomide, or a platin compound. 36.The method of claim 30, wherein radiotherapy comprises X-rayirradiation, UV-irradiation, γ-irradiation, or microwaves.
 37. Themethod of claim 15, wherein from about 10³ to about 10¹⁵ viral particlesare administered to the patient.
 38. The method of claim 37, whereinfrom about 10⁵ to about 10¹² viral particles are administered to thepatient.
 39. The method of claim 37, wherein from about 10⁷ to about10¹⁰ viral particles are administered to the patient.